KR20190052073A - Detection reagents and electrode arrays for multiple analyte diagnostic test elements, and methods of using it - Google Patents

Abstract

Detection reagents, multiple analyte test elements, test systems, and multiple assay methods are provided. In particular, the multi-analyte test element comprises (1) a first working electrode coated with a first analyte-specific reagent comprising an enzyme, a coenzyme and a first agent, and a first counter electrode pair, (2) And a second working electrode coated with a second analyte-specific reagent comprising a second mediator, wherein the second mediator is different from the first mediator. A single counter electrode may be used as the counter electrode for measurements of both the first and second analytes at each working electrode. In addition, mediator concentrations, measurement ranges, and applied potential differences are not the same for each analyte-specific measurement.

Description

Detection reagents and electrode arrays for multiple analyte diagnostic test elements, and methods of using it

Cross reference to related applications

The present application is related to U.S. Provisional Application No. (filed October 5, 2016). 62 / 404,258, which is hereby incorporated by reference as if fully set forth.

Technical field

This disclosure relates generally to chemical, engineering and medical / medical diagnostics, and more particularly to detection reagents and electrode arrays for multiple analyte diagnostic test elements, and multiple analytical methods using it.

Disposable diagnostic test elements have become commonplace to analyze ( i.e. , detect their presence and / or measure their concentration) of selected analytes in body fluid samples. For example, diabetic patients are typically involved in daily self-monitoring of at least blood glucose levels. If you do not take corrective action after determining your blood sugar concentration, you may need to take corrective action to bring your blood sugar concentration back into tolerance if your blood glucose concentration is too high or low, because it can have serious medical effects. Thus, daily self-monitoring of daily blood glucose levels is routine for diabetics, and the accuracy of such monitoring can mean a difference between life and death. Failure to maintain blood glucose levels within a regularly acceptable range can lead to severe diabetic complications, including, but not limited to, cardiovascular disease, kidney disease, nerve damage and blindness.

A number of assay systems are available, such as test instruments and associated diagnostic test elements, which enable a person to electrochemically or optically measure the glucose concentration in body fluids samples. In current test instruments, the information displayed after a successful blood glucose test is typically the individual blood glucose concentration shown in mg / dL or mmol / L (mM) and the date and time the measurement was performed. This information can be used in a variety of ways to prevent or reduce hyperglycemia in a short period of time in most cases, with the calculation of a planned / known carbohydrate intake and / or with knowledge of planned / known activities and / Is sufficient to allow adjustment or induction of ingestion and / or immediate administration of insulin. In addition, when the glucose concentration is low, diabetic patients can detect the need for sugar intake to avoid hypoglycemia.

If insulin is absent or insufficient, the body will not be able to use glucose as a fuel source to produce energy. When this happens, the body uses alternative fuel sources and breaks down fatty acids to produce energy, resulting in ketone by-products and increased ketone concentrations. Similarly, increased ketone levels in diabetic patients can lead to heart attacks, strokes, recreational drug use, or pathogenic diseases such as pneumonia, influenza, gastroenteritis, or urological infections.

Excessive ketone levels in diabetic patients can lead to diabetic ketoacidosis (DKA), a medical emergency that can lead to death if not treated. DKA prophylaxis can be achieved by measuring the ketone concentration and taking medical action if the ketone concentration rises above a certain threshold. The American Diabetes Association (ADA) recommends that patients with diabetes have a disease (such as a cold or flu) or have a ketamine concentration every 4-6 hours if the blood sugar level of a diabetic patient is greater than 240 mg / dL (Available at www.diabetes.org/living-with-diabetes/complications/ketoacidosis-dka.html).

Ketones are typically measured in urine and / or blood. However, in diabetics who undergo multiple blood glucose tests a day, performing separate urine and / or blood ketone tests in addition to the blood glucose test is time consuming and cumbersome. In addition, using separate tests to determine the ketone concentration requires additional diagnostic supplies and the associated costs, making it difficult to correlate glucose and ketone concentrations.

More recently, systems and methods have been developed to determine the concentration of both blood glucose and blood ketones in a single test with multiple analyte diagnostic test elements. However, in these multi-analyte test elements, blood glucose tests are completed more quickly than blood ketone tests, and the display of blood ketone concentration is delayed and thus provided after the blood glucose concentration. See, for example , U.S. Pat. 6,984,307. Alternatively, the concentrations of both blood glucose and blood ketones are delayed until the blood ketone test is complete.

In any case, especially if, in some cases, the blood ketone test can take nearly twice as long to complete the blood glucose test, waiting for one or two test results until the blood ketone test is complete is a relatively daily It can be quite burdensome and time consuming for a large number of diabetic patients. In addition, when the blood glucose concentration is provided separately before the blood ketone concentration, the test may be stopped before the blood ketone test is completed, or after the blood glucose test result is provided and before the result of the blood ketone test is appropriately considered, There is a possibility that the attention can be turned to the place.

Recent advances in multi-analyte testing are improved ketone reagent formulations that provide both the blood ketone and blood glucose concentrations within 7.5 seconds and even within seconds of each other after contacting the test element with the body fluid sample. For example, in International Patent Application Laid-Open Publication No. Hei. See WO 2014/068022. Another advancement of multi-analyte testing includes " ketone clock ", wherein the ketone clock is triggered when the blood glucose concentration is at a predetermined predetermined value and not only triggers the ketone trend analysis, but also the glucose and / If the value is exceeded, the blood ketone concentration is automatically provided to the blood ketone concentration. For example, in International Patent Application Laid-Open Publication No. Hei. See WO 2014/068024. Alternatively, if someone has a disease such as a cold or flu, a ketone watch can be started. See id .

However, current multi-analyte test elements require a complete detection reagent for each analyte of interest and a separate pair of working and counter electrodes for each analyte of interest.

Although the known methods and systems provide many advantages in terms of separately measuring glucose and ketone concentrations, there remains a need for additional systems and methods for simultaneously measuring glucose and ketone concentrations in the same diagnostic test element.

The concepts of the invention described herein include using a specific combination of the agents in a multi-analyte detection reagent such that a single counter electrode CE can be used with a plurality of analyte-specific working electrodes WE. The concept of the present invention is based on the fact that a second analyte-specific detection reagent having a first WE and a first CE pair coated with a first analyte-specific detection reagent comprising a first agent and a second analyte- RTI ID = 0.0 > WE < / RTI > In this manner, a single CE can be used as the CE for the first and second analyte measurements in each of the WEs. Further, the mediator concentration, the measuring range, the applied potential difference and the order in which these potential differences are applied to the sample may be different for each analyte-specific measurement. In other words, the concept of the present invention involves the use of detection reagents for at least two different analytes, wherein one detection reagent comprises a first agent that provides WE and CE functions for one analyte measurement , The same CE also provides a CE function for any other analyte measurement at different WEs with their analyte specific detection reagents, including an agent that is distinct from the first agent. Accordingly, the concepts of the present invention may be incorporated into exemplary dry detection reagents, multi-analyte diagnostic test elements, test systems, and multi-analyte measurement methods described herein and described in more detail below.

For example, detection reagents are provided for multi-analyte analysis comprising a first detection reagent for a first analyte of interest and a second detection reagent for a second analyte of interest.

The first detection reagent comprises the first enzyme-dependent enzyme or the substrate for the first enzyme, the first coenzyme, and the first medium. In some cases, the first coenzyme-dependent enzyme and the first coenzyme are attached, bound, integrated, or linked to each other.

The first enzyme-dependent enzyme may be an oxidase or dehydrogenase. In some cases, the first coenzyme dependent enzyme is selected from the group consisting of flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD) -or pyrroloquinoline-quinone (PQQ) -dependent dehydrogenase, especially FAD-, NAD- Or PQQ-dependent dehydrogenase as well as their enzymatically active mutants. In other cases, the first coenzyme-dependent enzyme is a glucose dehydrogenase, a glucose-6-phosphate dehydrogenase, or a glucose oxidase as well as an enzymatically active mutant thereof.

Similarly, the first coenzyme may be a compound according to Formula (I) or a salt thereof or a reduced form thereof, such as FAD, NAD, nicotinamide adenine dinucleotide phosphate (NADP), thio-NAD, thio-NADP, PQQ, . In some cases, the first coenzyme is FAD, NAD, NADP, or a compound according to formula (I) or a salt thereof or a reduced form thereof. In other cases, the first coenzyme is FAD.

The first mediator may also be selected from the group consisting of azo compounds or azo precursors, benzoquinone, melphalan blue, nitrosoaniline or nitrosoaniline precursors, phenazine or phenazine precursors, quinone or quinone derivatives, thiazine or thiazine derivatives, Complexes such as potassium ferricyanide and osmium derivatives, or combinations of phenazine / phenazine precursors and hexaamine ruthenium chloride and derivatives thereof. In some cases, the first agent is a nitrosoaniline derivative or a nitrosoaniline-based precursor, ferricyanide, ruthenium hexamine or phenazine. In other examples, the first agent is N, N-bis (hydroxyethyl) -3-methoxy-4-nitrosoaniline hydrochloride (referred to as BM 31.1144 or NA1144; Roche Diagnostics, Inc .; to be.

Thus, an exemplary first detection reagent is an FAD-dependent glucose dehydrogenase enzyme as an enzyme; FAD as coenzyme; And a nitrosoaniline-based precursor such as NA1144 as an intermediary.

The second detection reagent similarly comprises a second enzyme-dependent enzyme or substrate for a second enzyme, a second coenzyme, and a second agent, wherein the second agent can be distinguished from the first agent ( i.e. , It may not be the same as the first medium). In some cases, the second coenzyme-dependent enzyme and the second coenzyme are attached, bound, integrated, or linked to each other.

The second enzyme-dependent enzyme may be an oxidase or a dehydrogenase. In some cases, the second enzyme-dependent enzyme may be an enzymatically active mutant of FAD-, NAD- or PQQ-dependent dehydrogenase, particularly FAD-, NAD- or PQQ-dependent dehydrogenase as well as their enzymes. In other cases, the second coenzyme dependent enzyme is selected from the group consisting of an alcohol dehydrogenase, a glucose dehydrogenase, a glucose-6-phosphate dehydrogenase, a glucose oxidase, a glycerol dehydrogenase, a hydroxybutyrate dehydrogenase (HBDH), a malate dehydrogenase, Amino acid dehydrogenase, including dehydrogenase or L-amino acid dehydrogenase, as well as their enzymatically active mutants. In certain instances, the second enzyme-dependent enzyme is HBDH such as 3-HBDH as well as its enzymatically active mutant. Alternatively, the second coenzyme-dependent enzyme may be the same enzyme as the first coenzyme-dependent enzyme.

Similarly, the first coenzyme may be a compound according to Formula (I) or a salt thereof or a reduced form thereof, such as FAD, NAD, nicotinamide adenine dinucleotide phosphate (NADP), thio-NAD, thio-NADP, PQQ, . In some cases, the second coenzyme is thio-NAD, thio-NADP, or a compound according to formula (I), or a salt or an optionally reduced form thereof. In other cases, the second coenzyme is carba-NAD, carba-NADP, thio-NAD or thio-NADP.

The first mediator may also be selected from the group consisting of azo compounds or azo precursors, benzoquinone, melphalan blue, nitrosoaniline or nitrosoaniline precursors, phenazine or phenazine precursors, quinone or quinone derivatives, thiazine or thiazine derivatives, Complexes such as potassium ferricyanide and osmium derivatives, or combinations of phenazine / phenazine precursors and hexaamine ruthenium chloride and derivatives thereof. In some cases, the second mediator is a medol blue, a phenazine or phenazine precursor, or a quinone or quinone derivative. In other instances, the second agent is a phenazine derivative such as 1- (3-carboxy-propionylamino) -5-ethyl-phenazin-5-yumine (PG355).

When the first analyte is glucose, the second analyte can be an analyte associated with free fatty acids, ketones, glycerol or any other analyte representative of lipolysis, particularly free fatty acid metabolism such as ketones and ketone bodies. Thus, an exemplary second detection reagent is HBDH as an enzyme; Carba-NAD, carba-NADP, thio-NAD or thio-NADP as coenzymes; Lt; RTI ID = 0.0 > phenazine / phenazine < / RTI > precursors such as PG355. More specifically, the second detection reagent may be 3-HBDH, carba-NAD and PG355.

Alternatively, when the first and second analytes are the same analyte, such as glucose, the exemplary second detection reagent is an FAD-dependent glucose dehydrogenase enzyme as an enzyme; FAD as coenzyme; And pericyanide or nitrosoaniline other than NA1144 as an agent.

Also included is a multi-analyte diagnostic test element comprising a non-conductive base substrate having a first electrode system in communication with a first sensing reagent as described herein and a second electrode system in communication with a second sensing reagent as described herein Is provided. The first electrode system includes CE and WE pairs as well as associated conductive traces and contact pads. Likewise, the second electrode system includes WE as well as the associated conductive traces and contact pads. In some cases, additional electrode systems and detection reagents for other assays may also be included, provided that the additional detection reagent has a different agent than the agent in the first detection reagent. In other examples, the additional electrode systems also include sample-sufficient electrodes and / or integrity electrodes.

Also provided are systems comprising (1) a test meter configured to analyze a body fluid sample, and (2) one or more multi-analyte diagnostic test elements as described herein. The instrument is configured to receive the multiple analyte test elements and thus comprises a controller configured to determine a concentration of one or more analytes in the body fluid sample based on the response information obtained from the multiple analyte elements and to provide a test sequence. To help convey test results to the user, the instrument may also include one or more input devices and / or output devices.

In view of the foregoing, multiple analyte analysis methods may include applying or contacting the multiple analyte diagnostic test elements described herein to a body fluid sample; Applying an electrical test sequence to the body fluid sample to obtain response information about each analyte of interest; Determining a first analyte concentration in the sample from each response information for the test sequence, determining a second analyte concentration in the sample from each response information for the test sequence, and determining one or both analytes And displaying information on the concentration to the user. The methods may optionally include determining additional analyte concentrations when additional working electrodes and detection reagents are provided on the test element. The methods may optionally include modulating the therapy ( e.g., insulin) or modifying the diet based on one or more analyte concentrations. The methods also optionally include sending a message to at least one of a user, a healthcare provider, a caregiver, and a parent or guardian of the test element to adjust treatment or modify the diet based on one or more analyte concentrations .

In some cases, both analyte concentrations are displayed to the user: in other cases, only one analyte concentration is displayed, while the other analyte concentrations are displayed on one analyte, another analyte, or both analytes Is only displayed if a predetermined threshold or condition is met. In some cases, the first analyte is glucose and the second analyte is a ketone, wherein the glucose concentration is displayed to the user and the ketone concentration is displayed only when the predetermined threshold (s) or condition (s) are met And the predetermined threshold (s) or condition (s) may be a glucose concentration of about 240 mg / dL or a ketone concentration of about 0.6 mM to about 3.0 mM, or even about 0.6 mM to about 1.5 mM.

In other cases, the methods may include displaying a second analyte concentration, providing a warning, displaying a second analyte concentration, an action to be taken in response to the second analyte concentration exceeding a predetermined value (S) or condition (s) to at least one of providing a list, or sending a message to a user of the test element, a healthcare provider, a caregiver, and / or a parent or guardian And displaying it to the user when satisfied.

In summary, a number of analytes, including but not limited to amino acids, antibodies, bacteria, carbohydrates, drugs, lipids, markers, nucleic acids, peptides, proteins, toxins, viruses and other analytes as well as combinations thereof Detecting reagents, multiple analyte diagnostic test elements, test systems and multiple analytical measurement methods that can be used to determine concentration are provided.

These and other advantages, effects, features and objects of the inventive concept will become better understood from the following description. In the description, reference is made to the accompanying drawings, which form a part hereof and in which is shown by way of illustration and not limitation, embodiments of the inventive concept.

Advantages, effects, features and objects of the invention other than the above-mentioned developments will become more readily apparent when consideration is given to the following detailed description. This detailed description refers to the following drawings:
Figure 1 shows an exemplary test element configuration.
Figures 2A-2C illustrate exemplary electrode system configurations for multiple analyte test elements.
Figure 3 illustrates an exemplary test system that includes a meter and multiple analyte test elements as described herein.
Figures 4A-4F illustrate an exemplary electrical test sequence for multiple analyte measurements. Specifically, Figure 4A shows an exemplary test sequence with two direct current (DC) components (left panel) and an exemplary current response (right panel) therefor for multiple analyte measurements. Figure 4B shows an exemplary test sequence with two DC components (left panel) followed by a dormant component for multiple analyte measurements and an exemplary current response (right panel) therefor. Figure 4c shows an exemplary test sequence with a first dormant component, a first DC component, a second dormant component and a second DC component (left panel) and an exemplary current response thereto (right panel) for multiple analyte measurements Respectively. Figure 4c shows an exemplary test sequence with a first dormant component, a first DC component, a second dormant component and a second DC component (left panel) and an exemplary current response thereto (right panel) for multiple analyte measurements Respectively. FIG. 4E shows an exemplary embodiment of the present invention that includes an initial dormant component, an alternating current (AC) component, a pulsed first DC component, a pulsed second DC component different from the first DC component, and a third DC component ) And an exemplary current response thereto (right panel). FIG. 4F shows a schematic diagram of an embodiment of the present invention that includes a dormant component for multiple analyte measurements, an AC component, a pulsed first DC component, a pulsed second DC component different from the first DC component, and a pulse different from the first and second DC components (Left panel) and an exemplary current response (right panel) thereof.
Figure 5 shows a dose-response curve for an exemplary ketone detection reagent with mutant HBDH, high mediator content (PG355) and high molecular weight (Natrasol). Eight different levels of 3-hydroxybutyrate (3-HB) (0 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 3 mM, 4 mM and 8 mM) were tested.
Figures 6a and 6b show the results of crosstalk experiments in which a sample containing both 3-HB and glucose in varying concentrations is administered to the test elements. Specifically, Figure 6a shows the effect on glucose current in the presence of different levels of 3-HB (0 mM, 1 mM, 3 mM and 8 mM). Two glucose levels (0 mg / dL and 300 mg / dL) were tested, each with a different level of 3-HB. Figure 6b shows the effect on 3-HB current in the presence of different levels of glucose (0 mg / dL and 300 mg / dL). Four different levels of 3-HB (0 mM, 1 mM, 3 mM and 8 mM) were tested, each with different levels of glucose.
Figures 7a and 7b show dose-response curves for mutant HBDH, low mediator content (PG355), low polymer content, and other exemplary ketone detection reagents with high adjuvant content (NAD or cNAD). Specifically, FIG. 7a shows the dose-response curve of NAD and cNAD in which a sample containing various concentrations of 3-HB (0 mM, 1 mM, 2 mM, 3 mM and 4 mM) was administered to the test element . FIG. 7B shows a sample containing glucose at various concentrations (0 mg / dL, 57 mg / dL, 123 mg / dL, 520 mg / dL and 1000 mg / dL) in a test element having a ketone detection reagent and a glucose detection reagent Lt; / RTI > shows the dose-response curve of one NAD and cNAD.
Figure 8 shows the dose-response curve for multi-analyte test elements with an exemplary ketone detection reagent comprising a glucose detection reagent as well as a different agent (cPES) than the ketone detection reagent. The test elements contained various concentrations of 3-HB (0 mM, 0.25 mM, 0.5 mM, 1 mM, 1.25 mM, 1.5 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM and 8.0 mM) A sample is administered.
Figures 9A-9F illustrate the effect of different HBDH enzymes in exemplary ketone detection reagents. Specifically, Figure 9a shows the 3-HB current in the presence of glucose with wild-type HBDH; Figure 9b shows the 3-HB current in the presence of glucose with the AFDH3 HBDH mutant; Figure 9c shows the 3-HB current in the presence of glucose with the AFDH4 HBDH mutant; Figure 9d shows the glucose current in the presence of glucose with wild-type HBDH; Figure 9e shows the glucose current in the presence of glucose with the AFDH3 HBDH mutant; And Figure 9f shows the glucose current in the presence of glucose with the AFDH4 HBDH mutant.
Figures 10a and 10b show crosstalk experiment results for an alternative exemplary double detection reagent wherein a sample containing glucose (300 mg / dL) and 3-HB is injected from the side or top of the test element. Specifically, Figure 10a shows the effect on glucose current in the presence of different levels of 3-HB. Figure 6b shows the effect on the 3-HB current. Four different levels of 3-HB (0 mM, 0.5 mM, 1.5 mM and 8 mM) were tested, each with different levels of glucose.
11A and 11B show the results of crosstalk experiments in which ketone and glucose detection reagents were deposited through a slot-die coating instead of PicoJet® discrete dispensing, in which samples containing various concentrations of 3-HB and glucose were administered Respectively. Specifically, Figure 11a shows the effect on 3-HB current in the presence of different levels of glucose (0 mg / dL, 150 mg / dL and 300 mg / dL). Three different levels of 3-HB (0.5 mM, 1.5 mM, and 3 mM) were tested, each with different levels of glucose. Figure llb shows the effect on glucose current in the presence of different levels of glucose 3-HB (0.5 mM, 1.5 mM and 3 mM). Three glucose levels (0 mg / dL, 150 mg / dL, and 300 mg / dL) were tested, each with different levels of 3-HB.
Figures 12a and 12b illustrate a method of preparing a sample of the present invention by ink jet printing instead of PicoJet diacid dispensing or slot-die coating with a sample containing 350 mg / dL glucose, 350 mg / dL maltose, or 350 mg / dL xylose The results of the crosstalk experiments in which dual glucose detection reagents are deposited are shown. Specifically, FIG. 12A shows the reaction of an electrode having a detection reagent with mutant PQQ-GDH having low maltose sensitivity. No significant xylose response was observed in the mutant PQQ-GDH electrode. Figure 12B shows the reaction of an electrode with a detection reagent with FAD-GDH.
Corresponding reference characters indicate corresponding parts throughout the several views of the drawings.
The concept of the present invention permits various modifications and alternative forms, but its exemplary embodiments are illustrated by way of example in the drawings and are described in detail herein. It should be understood, however, that the description of the following exemplary embodiments is not intended to limit the inventive concept to the particular forms disclosed, but on the contrary, the intention is to cover at least the modifications and embodiments thereof as defined by the embodiments described herein and the following claims And is intended to cover all the advantages, effects, features, and objects falling within the scope of the claims. Accordingly, reference should be made to the embodiments described herein and the claims that follow to interpret the scope of the inventive concept. As such, it should be noted that the embodiments described herein may have advantages, effects, features, and objects that are useful in solving other problems.

Hereinafter, the detection reagents, the multi-analyte diagnostic test elements, the test systems, and the multi-analyte measurement methods will be described more fully with reference to the accompanying drawings, in which some embodiments are shown, will be. Indeed, detection reagents, multiple analyte diagnostic test elements, test systems, and multi-analyte measurement methods can be implemented in many different forms and should not be construed as limited to the embodiments described herein; Rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.

Likewise, many variations and other embodiments of detection reagents, multi-analyte diagnostic test elements, test systems, and multi-analyte measurement methods are well within the scope of the present invention, as well as other embodiments having the benefit of the teachings set forth in the foregoing description and the associated drawings. Will be apparent to one of ordinary skill in the art to which the disclosure relates. Accordingly, the detection reagents, the multi-analyte diagnostic test elements, the test systems, and the multi-analyte measurement methods are not limited to the specific embodiments disclosed, and variations and other embodiments are within the scope of the appended claims It shall be understood that such Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of detection reagents, multiple analyte diagnostic test elements, test systems and multiple analytical methods of measurement, Methods and materials are described herein.

In addition, a singular representation of an element by the indefinite article "a" or "an" means that more than one element is presented, unless the context clearly requires that only one and only one element is present It does not exclude possibilities. The indefinite article "a" or "an" thus usually means "at least one". Likewise, " comprise, " " comprise, " or " include, " or any grammatical variation are used in a non-exclusive manner. Thus, these terms may refer to both situations in which there are no further features in the entity described in this context, and situations in which one or more additional features exist, in addition to those introduced by these terms. For example, the expression " A has B ", " A comprises " and " A includes B " May refer to both situations in which one or more additional elements other than B, such as element C, elements C and D, or even additional elements, exist in A (i.e., a situation where A consists solely and exclusively of B) .

survey

A multi-analyte diagnostic test element based on the concept of the present invention, including using specific combinations of mediators in detection reagents for multiple analyte test elements so that a single CE can be used with multiple analyte-specific WEs, Test systems and multiple assay methods are provided. For example, one detection reagent with a first agent provides WE and CE functions for one analyte of interest, each having a different agent from the first agent ( i.e. , the first agent is the same as the subsequent agent ) Provides CE functionality to one or more other analytes of interest with their own detection reagent.

Detection reagents, multiple analyte diagnostic test elements, test systems, and multiple assay methods are useful in a variety of applications. For example, the multi-analyte diagnostic test elements may be used to monitor multiple analyte concentrations in a disease or disorder such as diabetes ( e.g., glucose and ketone) or heart disease ( e.g., cholesterol / lipid and glucose) . Similarly, multiple analyte diagnostic test elements can be used to monitor the progress of treatment or therapy, such as insulin therapy, in diabetes.

ller et al. (2000) Fresenius J. Anal. Chem. 366:560-568 및 미국 특허 출원 공개공보 No. 2009/0246808 를 참조할 수 있다. Detection reagents, diagnostic test elements and assay methods generally are described, for example, in Hnes et al. (2008) Diabetes Technol. Ther. 10: S10-S26, Haberm

ller et al. (2000) Fresenius J. Anal. Chem. 366: 560-568; 2009/0246808.

Although the disclosure relates to dual analyte detection reagents, diagnostic test elements, test systems and methods of measuring glucose and ketones, those skilled in the art will appreciate that other multi-analyte detection reagents, diagnostic test elements, test systems, and multi- Also, for example, a double test for glucose and 1,5-anhydroglucitol or HbA1c, a double test for glucose and cholesterol, a double test for glucose and lactate, or even a double test for glucose and fructosamine Will also be beneficial. It is also believed that two analytes can be measured through a single multi-analyte diagnostic test element. Thus, analytes of interest include, but are not limited to, alcohols, amino acids, 1,5-anhydroglucitol, cholesterol, fructosamine, glucose, glycerin, HbA1c, HDL ketone / ketone, lactate, lactate Dehydrogenase, malate, pyruvate, sorbitol, triglyceride, and uric acid. As used herein, " ketone " means a ketone such as acetoacetate and hydroxybutyrate (HB).

Advantageously, the detection reagents, multi-analyte diagnostic test elements, test systems, and multi-analyte measurement methods described herein provide information about multiple analytes that provide diagnostic information that is unique to a disease or disorder, such as diabetes, . Evaluations can range from detecting the presence of two or more analytes to determining the concentration of two or more analytes. Specifically, the systems and methods of the present invention allow people with diabetes to more easily comply with testing recommendations and safer therapies by simultaneously measuring, for example, glucose and ketone concentrations. In addition, the systems and methods of the present disclosure help a health care professional to initiate and / or modify therapy or treatment for a disease or disorder such as diabetes.

Detection reagent

Detection reagents may include a first analyte-specific detection reagent and a second analyte-specific detection reagent, but additional detection reagents are contemplated when three or more analytes are detected. As used herein, a "detection reagent" or "detection reagent" refers to a substance or chemical that changes at least one detectable property, particularly a physically and / or chemically detectable property, in the presence of at least one analyte ≪ / RTI > Typically, the property change occurs in the presence of at least one analyte to be detected, and does not occur in the presence of other substances. However, in practice, nonspecific property changes may be somewhat acceptable in the presence of other chemicals, and their presence in the sample of body fluids is almost impossible and / or exists only at very low concentrations.

Generally, the components of the first and second detection reagents are dissolved or suspended in the matrix such that the body fluid sample hydrates or dissolves the matrix, and the analyte of interest in the body fluid sample is diffused through the matrix, React with one or more active ingredients.

With respect to the first analyte-specific detection reagent, it may comprise at least one first coenzyme-dependent enzyme, at least one first coenzyme, and at least one first agent.

Thus, one component of the first analyte-specific detection reagent is the first enzyme-dependent enzyme. As used herein, " coenzyme-dependent enzyme " refers to an enzyme that requires an organic or inorganic co-factor termed coenzyme for catalytic activity.

In some cases, the first coenzyme-dependent enzyme may be a dehydrogenase. As used herein, " dehydrogenase " refers to an enzyme that catalyzes the oxidation of a substrate by transferring the hydride (H < ^- & gt ^; ) as a redox-equivalent, as a redox cofactor as described in elsewhere herein ≪ / RTI > Examples of dehydrogenases include, but are not limited to, alcohol dehydrogenase (EC 1.1.1.1 or 1.1.1.2), glucose dehydrogenase, glycerin dehydrogenase (EC 1.1.1.6), HBDH such as 3-HBDH (EC 1.1. 1.30) or beta-HBDH, alpha-HBDH and gamma-HBDH, lactate dehydrogenase (EC 1.1.1.27 or 1.1.1.28), L-amino acid dehydrogenase (EC 1.4.1.5), maleate dehydrogenase (EC 1.1. 1.37), or sorbitol dehydrogenase (EC 1.1.1.14), in particular NAD (P) / NAD (P) H-dependent dehydrogenase.

In some cases, the dehydrogenase is glucose dehydrogenase (GDH). Examples of GDH include, but are not limited to, glucose dehydrogenase (EC, 1.1.1.47), quinoprotein glucose dehydrogenase (EC, 1.1.5.2), such as pyrroloquinoline quinone (PQQ) dependent glucose dehydrogenase (EC 2.7.1.1), glucose-6-phosphate dehydrogenase (GDH-PQQ, also glucose-dye-oxidoreductase GlucDOR; see for example US Patent Nos. 7,749,437 and 9,017,544) (EC 1.1.1.49), nicotinamide adenine dinucleotide (NAD) dependent glucose dehydrogenase (EC 1.1.1.119) and flavin adenine dinucleotide (FAD) dependent glucose dehydrogenase (EC, 1.1.99.10) RTI ID = 0.0 > mutant < / RTI >

As used herein, a " mutated " or " mutant " coenzyme dependent enzyme refers to a genetically modified variant of a natural coenzyme dependent enzyme ( e. G., A wild type enzyme) And has an amino acid sequence that is different from the native enzyme-dependent enzyme in at least one amino acid. In general, mutant enzyme-dependent enzymes have increased heat and / or hydrolytic stability as compared to native enzyme-dependent enzymes.

Mutant enzyme-dependent enzymes can be obtained by mutating ( i.e. , replacing, adding or deleting) the native coenzyme-dependent enzyme from any biological source. As used herein, " biological source " means both prokaryotes and eukaryotes. The introduction of the mutation (s) can be localized or non-localized; However, in some cases, the localized mutation is due to recombinant methods known in the art in which at least one amino acid exchange is introduced into the amino acid sequence of the native enzyme. As such, site-specific or non-site-specific mutations can be introduced using recombinant methods known in the art, wherein, depending on the respective requirements and conditions, at least one of the amino acid exchanges is an amino acid It occurs within the sequence. In this regard, mutants may have increased thermal or hydrolytic stability as compared to wild type enzymes.

In some cases, the mutant coenzyme-dependent enzyme is a mutant glucose dehydrogenase (EC 1.1.1.47) or a mutant glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Examples of specific mutants GDH are described, for example, in International Patent Application Publication Nos. WO 2005/045016, WO 2009/103540, WO 2010/094632 and WO 2011/020856; And Baik et al. (2005) Appl. Environ. Microbiol. 71: 3285-3293 and Vasquez-Figueroa et al. (2007) ChemBioChem 8: 2295-2301. In particular, the GDH mutant may have a mutation at least at amino acid positions 96, 170 and / or 252. See, for example, International Patent Application Publication Nos. WO 2009/103540 and WO 2010/094632; And U.S. Patent Application Laid-Open Publication No. Hei. 2014/0322737. Specific amino acid substitutions are Glu96Gly, Glu170Arg, Glu170Lys and / or Lys252Leu, in particular Glu170Arg and Gln252Leu, in the glucose dehydrogenase of Bacillus subtilis . Another such mutant is PQQ-dependent GDH with improved substrate specificity as compared to wild-type counterparts ( e.g. improved glucose sensitivity with reduced or attenuated sensitivity to competing sugars such as maltose). For example, US Patent Nos. 7132270; 7,547,535 and 7,732,179.

Regardless of the mutation, the mutant has essentially the same activity as the native enzyme. The mutants of the natural enzymes described above must also be encoded by a nucleic acid molecule that encodes a natural enzyme and a nucleic acid molecule that is in a position to be hybridized under stringent hybridization conditions. As used herein, "stringent hybridization conditions" means that the nucleic acid to be hybridized is incubated in a treatment buffer (0.5 M NaPO4 (pH 7.15), 7% SDS; 1 mM EDTA) for about 12 hours at about 65 ° C, Followed by washing in buffer (40 mM NaPO4 (pH 7.15), 1% SDS; 1 mM EDTA) for about 30 minutes twice. One of the nucleic acids to be hybridized is immobilized and the other is provided with a detectable label. When the nucleic acids are hybridized to each other, the hybridization can be detected by a detectable label on a fixed nucleic acid. Methods for carrying out the hybridization reaction are known in the art.

Other suitable first coenzyme dependent enzymes include, but are not limited to, oxidizing enzymes such as aminotransferases such as aspartate or alanine aminotransferase, 5'-nucleotidase, cholesterol oxidase (EC 1.1.3.6) (EC 1.1.3.17), creatinine oxidase, glucose oxidase (EC 1.1.3.4; GOx) and lactate oxidase (EC 1.1.3.2; Lox) as well as their enzymatically active mutants .

In addition to the first enzyme-dependent enzyme, the first analyte-specific detection reagent comprises a first coenzyme, which may be a natural coenzyme or an artificial / stabilizing coenzyme. As used herein, the term "coenzyme" or "redox cofactor" is a hydride (H ^-) means a molecule that can act as the enzymatically the redox transition equivalents of water receptor, which is a substrate (such as For example , an analyte of interest) to an enzyme or coenzyme. As used herein, " redox equivalents " refers to the concepts commonly used in redox chemistry known to those skilled in the art. In particular, it relates to electrons transferred from the substrate of the coenzyme-dependent enzyme ( i.e. , the analyte of interest) to the coenzyme, or electrons transferred from the coenzyme to the electrode or indicator. Examples of coenzymes include, but are not limited to, FAD, NAD, NADP, PQQ, thio-NAD, thio-NADP, and compounds according to formula (I).

As mentioned elsewhere herein, the first coenzyme-dependent enzyme and the first coenzyme can be attached, bound, integrated or linked to each other. As such, they may not be physically discrete components of the detection reagent, but may instead be together in a single component ( e. G., A covalent bond or an ionically bound complex). Examples of such coenzyme-dependent enzymes / coenzymes include but are not limited to GOx (FAD coenzyme), FAD-dependent GDH (FAD-GDH), PQQ-dependent GDH, cholesterol oxidase (FAD coenzyme), and diaphorase Or FAD coenzyme).

It will be understood that the first coenzyme contained in the detection reagent of the present invention depends on the characteristics of the coenzyme-dependent enzyme. For example, PQQ can be combined with PQQ-dependent GDH, NAD can be combined with NAD-dependent GDH, and FAD can be combined with FAD-dependent GDH. NAD derivatives ( e.g., NAD / NADH and / or NADP / NADPH derivatives) include carba-NAD (cNAD). For example, in International Patent Application Laid-Open Publication No. Hei. See WO 2007/012494.

In some cases, the first coenzyme is an artificial / stabilized coenzyme. As used herein, an " artificial coenzyme " or " stabilized coenzyme " is chemically modified with respect to natural coenzyme and has a humidity at atmospheric pressure, a temperature in the range of about 0 캜 to about 50 캜, a pH in the range of about pH 4 to about pH 10 Means a coenzyme which has a higher stability than a natural coenzyme for a nucleophilic substance such as an acid and a base, and / or an alcohol or an amine, and which can exhibit its effect for a longer period of time as compared to a natural coenzyme under the same surrounding conditions do. In some cases, the artificial coenzyme has higher hydrolytic stability than the natural coenzyme, and the complete hydrolytic stability under test conditions is particularly advantageous. Similarly, the artificial coenzyme may have a lower binding constant than the original coenzyme of the coenzyme-dependent enzyme, for example the binding constant may be reduced by a factor of two or more.

As used herein, " about " is intended to include within a statistically significant range of values or values such as the stated concentration, length, width, height, angle, weight, molecular weight, pH, sequence identity, time frame, temperature, it means. Such a value or range may be within one digit of a given value or range, and may typically be within 20%, more typically within 10%, and even more typically within 5%. The permissible variations included in " about " will depend on the particular system under study and can be readily understood by those skilled in the art.

Examples of artificial coenzymes include artificial NAD (P) / NAD (P) H compounds, which are not limited to, but are natural derivatives of NAD / NADH or natural NADP / NADPH. In some cases, the artificial co-enzyme comprises a compound according to formula (I) as shown below, but not limited to:

Where:

A = adenine or an analogue thereof,

T = independently at each occurrence represents O or S,

U = independently for each occurrence represents OH, SH, BH ₃ ^- , or BCNH ₂ ^-

V = independently for each occurrence represents OH or phosphate group,

W is COOR, CON (R) ₂ , COR, or CSN (R) ₂ , wherein R in each case independently represents H or C ₁ -C ₂ -alkyl,

X _{1 and} X ₂ each independently represent O, CH ₂ , CHCH ₃ , C (CH ₃ ) ₂ , NH, or NCH ₃ ,

Y = NH, S, O, or CH ₂ ,

Z = optionally O, S and moieties comprising a cyclic having 5 C atoms including a heteroatom selected, and optionally one or more substituents from the N, and CR4 ₂ is during a residue CR4 ₂ coupled to a cyclic group and X ₂ ,

Where R4 = independently for each, is H, F, Cl or CH _3, but Z and pyridine moiety is not linked by a glycosidic bond,

Or a salt thereof or an optionally reduced form thereof.

Exemplary substituents on Z may be OH, F, Cl, and C ₁ -C ₂ alkyl, which is optionally fluorinated or chlorinated and / or OH- substituted OC ₁ -C ₂ alkyl.

Alternatively, the first residue V is OH and the second residue V is a phosphate group. Optionally, one OH group and one phosphate group may form a ring with the carbon atoms to which they are attached.

Examples of adenine analogs include, but are not limited to, C ₈ -substituted and N ₆ -substituted adenines, diaza modifications such as 7-diaza modifications such as 8-aza or combinations such as 7-diaza or 8 -aza or carboxylic analogs such as formate myth (where 7-diaza elastic element may be substituted by halogen in the 7-position), C ₁ -C ₆ - alkynyl, C ₁ -C ₆ - alkenyl or C ₁ -C ₆ - comprises the alkyl. Alternatively, the compounds include bicyclic, LNA and tricyclic diacids instead of 2-methoxy dioxyribose, 2'-fluorodioxy-ribose, hexitol, altritol or polycyclic analogues such as ribose. In one form of the (di) phosphate oxygen is also O ^- on for S ^- and / or BH ₃ ^- electronically such as, for O to NH, NCH ₃ and / or CH _2, and a = S for = O . ≪ / RTI > Also, at least one of the residues U in the compound according to formula (I) is different from OH and alternatively at least one residue U = BH ₃ ^- .

Alternatively, the artificial co-enzyme comprises a compound according to formula (I) as shown below, but not limited to:

here:

A = adenine,

T = independently for each occurrence O,

U = independently for each occurrence represents OH,

V = independently for each occurrence OH,

W = CON (R) ₂ , wherein R represents H,

X ₁ = O,

X ₂ = O,

Y = O, and

Z = a carbocyclic 5-membered ring of general formula (II):

And wherein a single bond is present between R ₅ 'and R ₅ ",

R ₄ = H,

R < ₅ > = CHOH,

R < ₅ > = CHOH,

R ₅ = CR 4 ₂ ,

R < ₆ > = CH, and

R ₆ '= CH.

Alternatively, the artificial coenzyme further comprises a compound according to formula (I) as shown below, but not limited to:

here:

A = adenine,

T = independently for each occurrence O,

U = independently for each occurrence represents OH,

V = OH in the first case, phosphate group in the second case,

W = CON (R) ₂ , wherein R represents H,

X ₁ = O,

X ₂ = O,

Y = O, and

Z = a carbocyclic 5-membered ring of general formula (II):

And wherein a single bond is present between R ₅ 'and R ₅ ",

R ₄ = H,

R < ₅ > = CHOH,

R < ₅ > = CHOH,

R ₅ = CR 4 ₂ ,

R < ₆ > = CH, and

R ₆ '= CH.

In some cases, the artificial co-enzyme may be carbazole-NAD or carbazole-NADP. Slama & Simmons (1988) Biochem. 27: 183-193; And Slama & Simmons (1989) Biochem. 28: 7688-7694. CarbANAd has the following structure:

Carba-NADP has the following structure:

Other compounds according to formula (I) include boranocarb-NAD, cyclopentyl-NAD and carba-NAD cyclophosphate. These compounds have the following structure:

For further details regarding the compounds according to formula (I) and their synthesis, see International Patent Application Publication Nos. WO 2007/012494; In addition, 7,553,615.

Other artificial coenzymes that can be used in the detection reagents described herein are described in International Patent Application Publication Nos. WO 1998/033936, WO 2001/049247, WO 2009/103540 and WO 2011/020856; U.S. Pat. 5,801,006; And Hutchinson et al. (1996) Chem. Commun. 24: 2765-2766.

In addition to the first enzyme-dependent enzyme and the first coenzyme, the first analyte-specific detection reagent comprises a first agent. As used herein, " mediator " means a chemical compound that increases the reactivity of the reduced coenzyme obtained by reaction with the analyte and transfers the electron to the electrode system or to a suitable optical indicator / optical indicator system it means.

The agent may be any species (generally electroactive) capable of participating in a reaction scheme involving an analyte, a coenzyme dependent enzyme, a coenzyme and its reaction product to produce a detectable electroactive reaction product. Typically, the involvement of an agent in a reaction is catalyzed by the oxidation of an analyte, a coenzyme-dependent enzyme, a coenzyme, or any one of the reaction products of one of them (for example, a coenzyme reacted with a different oxidation state) (E. G., Reduction) of the state. Various media exhibit suitable electrochemical behavior. The mediator can also be stable in oxidized form and optionally can exhibit reversible redox electrochemistry, exhibit good solubility in aqueous solutions, and can rapidly produce electroactive reaction products. A review of mediators that can be used to directly deliver redox equivalents to a suitable detection system and electrochemically determine blood glucose is described, for example, in Takaminami (2008) Mater. Integr. 21: 317-323 and Heller et al. (2008) Chem. Rev. 108: 2482-2505.

Examples of the first mediator include, but are not limited to, azo compounds or azo precursors, benzoquinone, melphalan blue, nitrosoaniline or nitrosoaniline precursors, thiazine or thiazine derivatives, transition metals such as potassium ferricyanide Complexes, ruthenium complexes such as ruthenium hexamine chloride, osmium derivatives, quinone or quinone derivatives, phenazine or phenazine precursors, and combinations of phenazine derivatives and hexaamine ruthenium chloride, and derivatives thereof. For example, in International Patent Application Laid-Open Publication No. Hei. WO 1998/035225; And United States Patent Nos. 5,286,362 and 8,008,037; And Gorton & Dominguez (2002) Rev. Mol. Biotechnol. 82: 371-392.

Examples of the azo compound and the azo precursor include, but are not limited to, 0.0 > 2014/0212903 < / RTI > especially azo compounds which do not form an azacyclic dimer.

Examples of nitrosoaniline compounds that can act as an intermediate precursor include, but are not limited to, those disclosed in European Patent Nos. 0620283 and 0831327; United States Patent Nos. 5,206,147 and 5,286,362; And international patent application publication no. WO < RTI ID = 0.0 > 2013131885. ≪ / RTI > In this manner, the nitrosoaniline-based agent precursor is decomposed into reversible mediator components upon contact with the body fluid sample.

Other examples of nitrosoaniline-based vehicle precursors include, but are not limited to, N- (2-hydroxyethyl) -N'-p-nitrosophenyl-piperazine, N, N-bis- P-nitroso-aniline, p-hydroxynitrosobenzene, N-methyl-N ', N'- - (4-nitrosophenyl) -piperazine, p-quinonedioxime, N, N-dimethyl-p- Phenyl) -morpholine, N-benzyl-N- (5'-carboxypentyl) -p-nitrosoaniline, N, N, N-bis- (2-hydroxyethyl) -3-chloro-4-nitrosoaniline, 2,4 N-bis- (2-methoxyethyl) -4-nitrosoaniline, 3-methoxy-4-nitrosophenol, N- (2-hydroxyethyl) -6 - Nitroso-1,2,3,4-tetrahydroquinoline N, N-bis (2-hydroxyethyl) -3-fluoro-4-nitrosoaniline, N, N, N-bis- (2-hydroxyethyl) -3-methylthio-4- nitrosoaniline, N- N- (2-hydroxyethyl) -N- (3-methoxy-2-hydroxy-isoquinolin- 1-propyl) -4-nitrosoaniline, N- (2- hydroxyethyl) -N- (3- (2-hydroxyethoxy) -2-hydroxy- , N- (2-hydroxyethyl) -N- (2- (2-hydroxyethoxy) -ethyl) -4-nitrosoaniline, and [(4-nitrosophenyl) imino] dimethanol- Chloride.

Examples of osmium derivatives include, but are not limited to, 1457572 and International Patent Application Laid-Open Publication No. Hei. 1998/035225.

Examples of phenazine or phenazine precursors include, but are not limited to, phenazine ethosulfate (PES), phenazine methosulfate (PMS), 1- (3- carboxypropoxy) -5-ethylphenazinium (CPES), 1- (3-carboxy-propionylamino) -5-ethyl-phena naphthalene- (PG355), or 1-methoxyphenazine methosulphate. For example, EP patent application publication no. 0.654.079; And Gorton (1986) Chem. Soc., Faraday Trans. 82: 1245-1258. In some cases, the phenazine may be a 1-amino-phenazine derivative. For example, in International Patent Application Laid-Open Publication No. Hei. See WO 2015/158645. In certain cases, the phenazine may be one of the following structures or derivatives thereof:

Examples of quinone or quinone derivatives include, but are not limited to, ortho and paraquinone, and quinone diimine. For example, Gorton (1986), supra ; Degrand & Miller (1980) J. Am. Chem. Soc. 102: 5728-5732; Kitani & Miller (1981) J. Am. Chem. Soc. 103: 3595-3597; And Baldwin (1983) Anal. Chem. 55: 1588-1591.

In some cases, the first agent is N, N-bis (hydroxyethyl) -3-methoxy-4-nitrosoaniline hydrochloride (NA1144).

Regarding the concentration of the first mediator, the first mediator is generally a very stable mediator ( i.e. , at least more stable than the second mediator) and is provided at a higher concentration than the second mediator ( i.e. , Lt; / RTI > However, those skilled in the art will understand that the relative mediator concentration is determined in part by the analyte concentration range expected to be detected. For example, when monitoring glucose and ketones, the 3-HB concentration range is significantly less than the glucose concentration range. Thus, the ketone agent may be a lower concentration than the glucose vehicle. More specifically, a major consideration for glucose mediator concentration ( i . E., The first mediator) is that the glucose mediator concentration is high enough to support the signal in the high glucose range, thereby providing sufficient oxidation medium For example, nitroso form and quinonediamine form. Depending on the reagent formulation and on the glucose-only test element, the first agent concentration can be from about 10 mM to about 40 mM, from about 15 mM to about 35 mM, from about 20 mM to about 30 mM, or about 25 mM have. Alternatively, the first mediator concentration may be about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, or about 40 mM. Alternatively, the first agent concentration may be about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, about About 30 mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, or about 40 mM.

When the coenzyme is NAD / NADH, the mediator may be a quinone (e.g., ortho or paraquinone), quinone diimine, phenazine, phenoxazine, or phenothiazine.

Similarly, when the coenzyme is cNAD / cNADH, the medium can be PG355, cPES, PES, PMS or N, N-bis (hydroxyethyl) -3-methoxy-4-nitrosoaniline hydrochloride.

Additional non-limiting examples of reagent materials that can operate to detect the presence and / or concentration of glucose are disclosed in U.S. Patent Application Publication Nos. 2003/0146113 and 2014/0212903; And United States Patent Nos. 7,727,467 and 8,008,037, and Huang et al. (2014) Clin. Chim. Acta. 433: 28-33.

With respect to the second analyte-specific detection reagent, it may comprise at least one second coenzyme-dependent enzyme, at least one second coenzyme, and at least one second agent, wherein at least one second agent Is distinguished from at least one first agent included in the first analyte-specific detection reagent. Generally, the components of the first and second detection reagents are dissolved or suspended in the matrix such that the body fluid sample hydrates or dissolves the matrix, and the analyte of interest in the body fluid sample is diffused through the matrix, React with one or more active ingredients.

The second enzyme-dependent enzyme may be one of the enzymes listed above and may be the same enzyme as the first enzyme-dependent enzyme when performing duplicate analyte measurements in the same diagnostic test element. Alternatively, the first and second enzyme-dependent enzymes are not the same.

As noted elsewhere herein, the second coenzyme-dependent enzyme and the second coenzyme can be attached, bound, integrated or linked to each other. As such, they may not be physically discrete components of the detection reagent, but may instead be together in a single component ( e. G., A covalent bond or an ionically bound complex).

In some cases, the second coenzyme dependent enzyme is HBDH. Examples of HBDH include, but are not limited to, alpha-HBDH, beta or 3-HBDH, and gamma-HBDH.

In addition to the second enzyme-dependent enzyme, the second analyte-specific detection reagent may comprise a second coenzyme, which may be a natural coenzyme or an artificial / stabilizing coenzyme. The second coenzyme may be the coenzyme listed above and may even be the same coenzyme as the first coenzyme. Alternatively, the first and second enzyme-dependent enzymes are not the same.

As described above for the first analyte-specific detection reagent, examples of the second coenzyme include but are not limited to FAD, NAD, NADP, PQQ, thio-NAD, thio-NADP and the compound according to formula (I) . In some cases, such as where the second analyte is a ketone, the second coenzyme is selected from the group consisting of thio-NAD, thio-NADP or a compound according to formula (I) or a salt or an optionally reduced form thereof, Bar-NADP.

In addition to the second enzyme-dependent enzyme and the second enzyme, the second analyte-specific detection reagent comprises a second agent. As described above for the first analyte-specific detection reagent, examples of the second mediator include, but are not limited to, azo compounds or azo precursors, benzoquinone, melphalan blue, nitrosoaniline or nitrosoaniline precursors, Combinations of thiazine or thiazine derivatives, transition metal complexes such as potassium ferricyanide, ruthenium hexamine chloride and osmium derivatives, quinone or quinone derivatives, phenazine or phenazine precursors, and phenazine derivatives and hexaamine ruthenium chloride, and And derivatives thereof, with the proviso that the second agent is not the same as the first agent. In some cases, such as where the second analyte is a ketone, the second agent may be a 1-amino-phenazine derivative such as a phenazine or a phenazine derivative, especially PG355. For example, in International Patent Application Laid-Open Publication No. Hei. See WO 2015/057933.

Regarding the concentration of the first mediator, the first mediator is generally a very stable mediator ( i.e. , at least more stable than the second mediator) and is provided at a higher concentration than the second mediator ( i.e. , Lt; / RTI > As noted above, one of ordinary skill in the art understands that the relative mediator concentration is determined in part by the analyte concentration range expected to be detected. For example, when monitoring glucose and ketones, the 3-HB concentration range is significantly less than the glucose concentration range. Thus, the ketone agent may be a lower concentration than the glucose vehicle. More specifically, with respect to the "low" and "high" concentrations of the second agent, the concentration may be from about 2.5 mM to about 8.5 mM, from about 3.0 mM to about 7.5 mM, from about 3.5 mM to about 7.0 mM, About 6.5 mM, about 4.5 mM to about 6.0 mM, or about 5.0 mM to about 5.5 mM. Alternatively, the second vehicle concentration may be about 2.5 mM, about 3.0 mM, about 3.5 mM, about 4.0 mM, about 4.5 mM, about 5.0 mM, about 5.5 mM, about 6.0 mM, about 6.5 mM, mM, about 8.0 mM, or about 8.5 mM.

In other words, the ratio of the second agent to the first agent is about 1: 1.5, about 1: 2, about 1: 2.5, about 1: 3, about 1: 3.5, about 1: 4, about 1: : About 1: 5, about 1: 5.5, about 1: 6, about 1: 6.5, about 1: 7, about 1: 7.5, about 1: 8, about 1: 1:10. ≪ / RTI > An exemplary ratio can be 1: 1.6 ( e.g. , 7.5 mM PG355: 16 mM NA1144) to about 1: 8 (5 mM PG355: 40 mM NA1144).

Additional non-limiting examples of reagent materials operable to detect the presence and / or concentration of ketones are described in U.S. Pat. 8,920,628 and 8,921,061.

In addition to the coenzyme-dependent enzymes, coenzymes, and mediators, the detection reagent may include other materials used in qualitative and / or quantitative analysis of the analyte of interest. For example, detection reagents may include various adjuvants to enhance their various properties or properties. See, for example , U.S. Pat. 7,749,437. In addition, the detection reagent may comprise a material for facilitating their placement on each substrate and for improving its adhesion, or a material for increasing the rate of hydration of the reagent materials by the sample fluid. In addition, the detection reagent may comprise a component that improves the physical properties of the resulting dried reagent layer and enhances the absorption of body fluid samples for analysis. For example, in International Patent Application Laid-Open Publication No. Hei. See WO 2013131885.

Examples of adjuvant materials include, but are not limited to, buffers, carrier materials, colorants, compatible solutes, deliquescent materials, detergents, fillers, film formers, film openers, gellants, pigments, solid particles, stabilizers, , Thixotropic agents, and viscosity modifiers.

Examples of buffers include, but are not limited to, phosphate buffered saline, Tris buffer, citrate buffer, glycerine phosphate buffer, and Good Buffers. A further detailed description of the buffer in the detection reagent can be found in, for example, International Patent Application Publication No. < RTI ID = 0.0 > No. < / RTI & It can be found at 2012/010308.

Examples of deliquescent materials include, but are not limited to, sodium chloride, calcium chloride, magnesium chloride, zinc chloride, potassium carbonate, potassium phosphate, canelite, ammonium iron citrate, potassium hydroxide, and sodium hydroxide. A further detailed description of the buffer in the detection reagent can be found in, for example, International Patent Application Publication No. < RTI ID = 0.0 > No. < / RTI & 2014/037372.

An example of a detergent that may be included in the detection reagents include, but are not limited to, ammonium salts, as well as water-soluble soap and water-soluble synthetic surface-active compounds, such as alkali, soil alkali or an optionally substituted higher fatty acids (e.g., oleic acid or Stearic acid), mixtures of natural fatty acids ( e. G. , Coconut or tallow oil), fatty acid sulfates, sulfonic acid esters, salts of alkylsulfonic acid taurine salts of fatty acids, fatty acid amides and ester amides. Examples of additional detergents include ester amide sodium-N-methyl-N-oleoyltaurate, N-octanoyl-N-methyl-glucamide, mega 8 (N-methyl-N-octanoylglucamide, (DONS), RHODAPEX® (especially CO-433 or CO-436), TEGO® Wet 265 (Evonik Resource Efficiency GmbH, Essen, Germany), TERGITOL® 15-s-19 (Dow Chemical Corp.; Midland, MI USA) and the N-methyl oleyl taurate sodium salt, a fatty acid salt sold under the trade name GEROPON® T77 (Rhodia HPCII (Home, Personal Care & Industrial Ingredients).

Examples of film formers and thixotropic agents include, but are not limited to, polyvinyl propionate dispersions, polyvinyl esters, polyvinyl acetate, polyacrylic esters, polymethacrylic acid, polyvinyl amides, polyamides, polystyrenes, Butadiene, styrene or maleic acid esters. One more specific thixotropic agent includes silica sold under the trade name KIESELSAURE SIPEMATE FK 320 DS (Degussa AG), while more specific film formers include polyvinyl pyrrolidone sold under the trade name POLYVIDYLOLIDON KOLLIDON 25 (BASF) (PVP) and polyvinyl propionate dispersions.

Examples of solid particles include, but are not limited to, silicon dioxide, sodium silicate or aluminum silicate, silica particles such as diatomaceous earth, metal oxides such as titanium oxide and / or aluminum oxide, nanoparticles of silicon dioxide, aluminum oxide or titanium oxide Synthetic oxides such as nanoparticles of oxidizing materials such as kaolin, powdered glass, amorphous silica, calcium sulfate and barium sulfate.

Examples of stabilizers for coenzyme-dependent enzymes include, but are not limited to, polysaccharides and mono- or di-fatty acid salts. Other stabilizers include trehalose and sodium succinate sold under the trade name D - (+) - trehalose dihydrate (Sigma Chemical Co).

Examples of swelling agents include, but are not limited to, methyl vinyl ether maleic anhydride copolymer, xanthan gum, and methyl vinyl ether maleic acid copolymer. Examples of thickeners include, but are not limited to, starches, gums ( such as pectin, guar gum, locust bean gum (carob seed) gum, konjac gum, xanthan gum, alginates and agar), casein, Pico colloid; Cellulose and semi-synthetic cellulose derivatives (carboxymethyl-cellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, methylhydroxyethylcellulose); Polyvinyl alcohol and carboxy-vinylate; And bentonites, silicates, and colloidal silicas. More specific forms of thickeners include xanthan gum sold under the trade name Keltrol ^® F (CP Kelco US, Inc.) and carboxymethylcellulose sold under the trade name AQUALON CMC 7F PH (Hercules Inc., Aqualon Division).

Additional details regarding the detection reagents and their components that can be used herein are described, for example, in Hones et al. (2008), supra , International patent application publication Nos. WO 2007/012494, WO 2009/103540, WO 2010/094426, WO 2010/094427, WO 2011/012269, WO 2011/012270 and WO 2011/012271. For test materials that may be used herein, reference is made to EP Patent Application Publication Nos. 0354441, 0431456, 0302287, 0547710 and 1593434 can be further referred to.

Detection reagents are generally described as being used in electrochemical test elements, however, the detection reagents herein may also be used in optical test elements. In this case, the detection reagent may also include an indicator. As used herein, an " indicator " is influenced by the process of analyte detection, particularly the detection reaction of an enzymatic reaction, so that at least one property change of the indicator can be recorded in the course of the detection reaction , ≪ / RTI > any desired substance. In some cases, this attribute may be an optical property. Thus, the indicator may be at least one dye. A further detailed description of the buffer in the detection reagent can be found in, for example, International Patent Application Publication No. < RTI ID = 0.0 > No. < / RTI & 2014/0363835.

As an optical indicator or optical indicator system, any desired substance that is reducible and undergoes a detectable change in optical properties, such as, for example, color, fluorescence, reflectance, transmittance, polarization and / or index of refraction during reduction, can be used. Determination of the presence and / or amount of the analyte in the sample can be performed by the naked eye and / or by a detection device using a photometric method suitable for the person skilled in the art. In some cases, heteropoly acids such as 2,18-phosphomolybdic acid are used as optical indicators, which are reduced to the corresponding heteropoly blue.

Also, the use of fluorescent agents to detect glucose concentrations in diagnostic test elements is described, for example, in EP Patent No. 1780288 and International Patent Application Laid-Open Publication No. Hei. WO 2009/015870. Glucose-induced changes in fluorescence of proteins and other phosphors are also known. Pickup et al. (2005) Biosens. Bioelectron. 20: 2555-2565, and U.S. Patent Application Laid-Open Publication No. Hei. 2012/0053429.

It should be understood that the chemical of the reaction scheme of the detection reagent herein may be selected in light of various chemical factors related to the system, including the identity of the analyte and the sample material. Nevertheless, for a given analyte or substance, a variety of different reactive components may be present in the catalyst (often a variety of enzymes will be useful), a co-reactant ( e.g. , various mediators may be useful) Diversity may be useful). Many such detection reagents and their reactive components and reaction products are known, and examples of several different enzymes include those listed in Table 1.

In view of the above, exemplary dual detection reagents for multiple analyte analysis can include, but are not limited to those listed in Table 2.

Additional detection reagents that can be combined with one of the above described detection reagents include, but are not limited to: (1) a lactate detection reagent wherein LDH is a coenzyme dependent enzyme, Carba -NAD is a coenzyme and PG355 is mediated; (2) a lactate detection reagent wherein the lactate oxidase (LOx) is a coenzyme-dependent enzyme, the flavone mononucleotide is a coenzyme, and NA1144 is a mediator; (3) Fructosamine detection reagent wherein fructosamine oxidase (FOx) is a coenzyme-dependent enzyme, FAD is coenzyme, and NA1144 is a mediator; (4) Cholesterol oxidase (CHOx) is a coenzyme dependent enzyme, FAD is a coenzyme, NA1144 is a medium, cholesterol detection reagent; (5) a choline detection reagent wherein choline oxidase (COx) is a coenzyme-dependent enzyme, FAD is a coenzyme, and NA1144 is a mediator.

Multiple analyte diagnostic test elements

The multiple analyte diagnostic test elements may include an electrode system having at least two WEs and at least one CE in conjunction with other electrode system components disposed on the substrate. The aforementioned detection reagents are incorporated into the dry film detection reagent matrix provided on the insert support substrate or the base for the test element and are used to electrochemically analyze the body fluid sample for the presence or concentration of one or more analytes of interest And is in physical and / or electrical contact with the system.

Diagnostic test elements are generally known and are available in different forms that may be applied throughout this disclosure. For example, test strips, test tapes, test discs, types of foldable test elements ( e.g. , according to the Leporello principle), and other types of test elements known to those skilled in the art. Hereinafter, the concept of the present invention will be described substantially with reference to test elements such as test strips, but it is understood that other embodiments are also possible and within the scope of the present disclosure.

Diagnostic test elements are typically provided in the form of a disposable test strip having a layered structure comprising a nonconductive base substrate, a spacer and a cover. Additional details of a test element comprising a similar layered structure can be found in U.S. Patent Nos. 7,727,467 and 8,992,750. In this manner, the test elements may be any one of a plurality of rolls of material, sheets of material, or any other material stock made in accordance with the principles of the present disclosure. Generally, the material selection for making the test elements includes any material that is sufficiently flexible for roll processing, but is strong enough to impart useful stiffness to the finished test elements. In addition, the test elements may include one or more graphics to provide guidance to the user on proper handling and use.

In view of this, one part of the diagnostic test element herein is a base or support substrate on which the various components can be constructed, deposited and / or deployed. The substrate includes a first surface facing the spacer and a second surface facing the first surface. The substrate also has opposed first and second ends ( e.g. , a respective dosing end and meter insertion end) and opposing side edges extending between the first and second ends. In some cases, the first and second ends of the substrate and the opposing side edges form a generally rectangular shape; Any one of a number of forms that allow the test elements to function as described herein is also contemplated.

Typically, the substrate is made of, but is not limited to, a flexible polymer comprising a polyester or polyimide such as polyethylene naphthalate (PEN) or polyethylene terephthalate (PET). Alternatively, the substrate may be made of any other suitable material that allows the substrate to function as described herein.

In addition to the substrate, the diagnostic test elements herein may include a spacer disposed on a first surface of the substrate, wherein the spacer includes at least one edge defining a boundary of the capillary channel formed between the cover and the substrate. As with the substrate, the spacer may be made of an insulating material, such as a flexible polymer, including, for example, an adhesive coated PET. In some cases, the spacer may be a PET film coated on both sides with a tackifier. Thus, the spacer includes one surface coupled to the first surface of the substrate using any one or combination of a wide variety of commercially available adhesives. Alternatively, the substrate may be coupled to the spacer by welding, such as thermal or ultrasonic welding. However, in some cases, if the cover and / or substrate are dimensioned to function as spacers, the spacers may be omitted.

In addition to the substrate and spacers, the diagnostic test elements herein may comprise a cover positioned over the spacer. The cover is generally sized and shaped to match the substrate and thus extends between opposite side edges of the substrate and extends to the first and second ends of the substrate. Alternatively, one of the cover or substrate is beyond the change to the test element to be (that is, the test elements are, including the overhang / cantilever in the sampling end portion) that allows to function as the distance predefined described herein Can be extended. As a result, the capillary sample chamber is defined as the space between the substrate and the cover bounded by one or more edges of the spacer.

Like the substrate and the spacer, the cover can be made of an insulating material such as a flexible polymer, for example, comprising an adhesive coated PET. One particular non-limiting example of a suitable material comprises a transparent or translucent PET film. Thus, the cover includes a lower surface that can be coupled to the spacer using any one or combination of a wide variety of commercially available adhesives. Alternatively, the cover may be coupled to the spacer by welding, such as heat or ultrasonic welding.

The substrate, the substrate, the spacer, and the cover together form a sample chamber in which a portion of the electrode system and the detection reagents are accessible to the body fluid sample for measurement. In some cases, the test elements may have a full width end dose (" FWED ") that allows the sample to fill the sample chamber at or near the first end ( i.e. , front) There are capillary channels on the side) are the test elements. In the FWED test elements, the spacer extends between opposite side edges of the substrate to form a sample chamber at a portion of the cover. Spacers are contemplated to be made of a single component or even a plurality of components. Nevertheless, the spacers define the boundaries of the sample chamber by extending across the entire width of the substrate, including the edge edges that are substantially parallel to the first end of the substrate and face the ends thereof.

It is further contemplated that the sample chamber may be a conventional capillary channel ( i . E., Defined on more than one side). In this manner, the end edge of the spacer includes a plurality of portions positioned between the first and second ends of the substrate and the opposite side edge to define a boundary of the sample chamber having the sample inlet at the first end of the test elements A U-shaped pattern can be formed. Other suitable embodiments contemplate the end edges of spacers that form capillary channels of semi-oval, semicircular or other shape, and one or more portions of the end edges may include linear or non-linear edges along all or part of their length have.

As described above, in some cases, the spacers may be omitted, and the sample chamber may be limited only by the substrate and the cover. See, for example , U.S. Pat. 8,992,750.

Additionally or alternatively, to use capillary action, the sample chamber may be pressurized, such as by applying pressure to the sample fluid to push it into the sample chamber and / or creating a vacuum on the sample chamber to pull the body sample fluid It can be increased by other means. In addition, one or more surfaces of the sample chamber may be formed of a hydrophilic material, provided with a coating of hydrophilic material, or subjected to a hydrophilic enhancement treatment to facilitate filling the sample chamber with a bodily fluid sample.

For example, the sample chamber may comprise an adsorbent material. Examples of adsorbent materials include, but are not limited to, cellulose derivatives such as polyester, nylon, cellulose, and nitrocellulose. If included, the adsorbent material facilitates ingesting fluid sample fluid by helping to wick fluid into the sample chamber. The use of an adsorbent material also serves to further reduce the pore volume of the sample chamber receiving the body fluid sample fluid.

Figure 1 is a perspective view of an exemplary diagnostic test element 10. In an exemplary embodiment, the test element 10 includes a non-conductive support substrate 12, an electrical conductor (not shown) formed on the support substrate 12 defining a plurality of electrode traces (not shown) And a cover (16) positioned on the substrate (14). In some cases, the electrical conductor may form any number of electrode traces, electrodes, and contact pads that allow the test element 10 to function as described herein and are described in more detail below.

1, the diagnostic test element 10 may have a substantially rectangular shape, but may be any of a number of forms that allow the test element 10 to function as described herein One is also considered. In addition, the test element 10 may be any one of a plurality of rolls of material, sheets of material, or any other material stock made in accordance with the principles of this disclosure. In general, the material selection for manufacturing the test element 10 includes any material that is sufficiently flexible for roll processing, but is strong enough to impart useful stiffness to the completed test element 10. [

In an exemplary embodiment, the support substrate 12 of the diagnostic test element 10 includes a first surface 18 facing the spacer 14 and a second surface 20 facing the first surface 18 do. The support substrate 12 also has opposed first and second ends 22 and 24 and opposing side edges 26 and 28 extending between the first and second ends 22 and 24. In some cases, the first and second ends 22, 24 and opposing side edges 26, 28 of the support substrate 12 generally form a rectangular shape. Alternatively, the first and second ends 22, 24 and opposite side edges 26, 28 may be of any of a variety of shapes and sizes to enable the test element 10 to function as described herein . In some cases, the support substrate 12 may be made of a flexible polymer including, but not limited to, a polyester or polyimide such as polyethylene naphthalate (PEN). Alternatively, the support substrate 12 may be made of any other suitable material that allows the support substrate 12 to function as described herein.

An electrical conductor is provided that forms an electrode trace on the first surface (18) of the support substrate (12). The electrical conductors may be aluminum, carbon ( e.g., graphite), cobalt, copper, gallium, gold, indium, iridium, iron, lead, magnesium, mercury (as amalgam), nickel, niobium, osmium, But are not limited to, materials including, but not limited to, platinum, rhodium, selenium, silicon (e.g., heavily doped polycrystalline silicon), silver, tantalum, tin, titanium, tungsten, uranium, vanadium, zinc, zirconium, . In some cases, the electrode traces are insulated from the remainder of the electrical conductor by laser ablation or laser scribing, all of which are well known in the art. In this way, the electrode traces can be manufactured broadly, for example, by field ablation, or at least by removing electrical conductors from regions extending around the electrodes by, for example, line scribing. Alternatively, the electrode traces may be fabricated by other techniques such as, for example, stacking, screen printing, photolithography, and the like.

In an exemplary embodiment, the diagnostic test element 10 is an FWED test element having an inlet or capillary channel 30 at a first end 22 of the support substrate. It is further contemplated that the sample chamber may be a conventional capillary channel ( i . E., Defined on more than one side). In the FWED test element, the spacer 14 extends between the opposite side edges 26, 28 of the support substrate 12 to form a capillary channel 30 in the cover and portion. It is contemplated that the spacer 14 may be made as a single component or even as a plurality of components. Nevertheless, the spacer 14 must include an end edge 32 that is substantially parallel to and opposite the end of the support substrate 12, thereby extending across the entire width of the support substrate 12 Thereby defining the boundaries of the capillary channel 30. Alternatively and as noted above, the end edge 32 includes multiple portions located between the first and second ends 22, 24 and opposite side edges 26, 28 of the support substrate 12 To form a generally U-shaped pattern defining the boundary of the capillary channel 30 having the sample inlet at the first end 22 of the test element (not shown). Other suitable embodiments contemplate the end edges of spacers that form capillary channels of semi-oval, semicircular or other shape, and one or more portions of the end edges may include linear or non-linear edges along all or part of their length have.

The spacers 14 are made of an insulating material such as, for example, a flexible polymer including an adhesive coated polyethylene terephthalate (PET) -bonded polyester. One specific non-limiting example of a suitable material comprises a white PET film, both sides of which are coated with a tackifier. The spacer 14 may be constructed from a variety of materials and may include an inner surface 34 that can be coupled to the first surface 18 of the support substrate 12 using any one or a combination of a wide variety of commercially available adhesives, . Also, when the first surface 18 of the support substrate 12 is exposed and not covered by the electrical conductor, the cover 16 is coupled to support the substrate 12 by welding, such as heat or ultrasonic welding . In addition, the first surface 18 of the support element 12 may be printed with a product labeling or command (not shown), for example, for use with the test element 10.

The cover 16 extends between the opposing side edges 26 and 28 of the support substrate 12 and extends to the first end 22 of the support substrate 12. In the exemplary embodiment, Alternatively, the cover 16 may extend beyond the first end 22 by a predetermined distance to allow the test element 10 to function as described herein. Thus, in an exemplary embodiment, the capillary channel 30 includes a cover 16 and a support substrate 12, bounded by a first end 22 and opposite side edges 26,28 of the support substrate 12. [ And the end edges 32 of the spacers 14. As shown in Fig.

The cover 16 may be made of an insulating material such as, for example, a flexible polymer comprising an adhesive coated PET-polyester. One particular non-limiting example of a suitable material comprises a transparent or translucent PET film. The cover 16 can be constructed of a variety of materials and includes an inner surface 36 that can be coupled to the spacer 14 using any one or a combination of various commercially available adhesives. Alternatively, the cover 16 may be coupled to the spacer 14 by welding, such as by heat or ultrasonic welding.

The diagnostic test elements include a CE / WE electrode pair, at least one individual WE, and at least one electrically conductive pathway of the electrically conductive material provided on the first surface of the support such that, for example, the electrode systems are coplanar, System. It is contemplated, however, that electrode systems can be formed on opposite surfaces such that one electrode system is on the first surface of the support and the other electrode system is on the opposite surface of the cover. See, for example , U.S. Pat. 8,920,628. Nonetheless, the electrically conductive material is typically arranged on the substrate in a manner that provides one or more electrically conductive paths. Specific arrangements of electrically conductive materials may be provided using a number of techniques including chemical vapor deposition, laser ablation, lamination, screen printing, photolithography, and combinations of these and other techniques. One particular method for removing portions of conductive material is described, for example, in U.S. Pat. Laser ablation or laser scribing, and more particularly, extensive field laser ablation as disclosed in U.S. Pat. No. 7,073,246. In this way, the electrode system can be manufactured broadly, for example by removing the electrically conductive material from the substrate by wide field ablation, or at least by, for example, line scribing. Alternatively, the electrode system may be fabricated by other techniques such as, for example, lamination, screen printing, photolithography, and the like.

인 임의의 원하는 두께일 수 있다.Briefly, laser ablative techniques are commonly used to fabricate metal layers or multilayer compositions, including insulating materials and conductive materials ( e.g., metal-laminates of metal layers coated on insulating materials or laminated to insulating materials) Including ablation of conductive materials). The metal layer may comprise a pure metal, an alloy or other material that is a metal conductor. Examples of metal or metal-like conductors include but are not limited to aluminum, carbon (e.g., graphite and / or graphene), copper, gold, indium, nickel, palladium, platinum, silver, titanium, And alloys or solid solutions of these materials. In one embodiment, the materials are selected to be essentially non-reactive to the biological system, including, but not limited to, gold, platinum, palladium, carbon and iridium tin oxide. The metal layer can be about 500 < RTI ID = 0.0 >

Lt; / RTI > may be any desired thickness.

In connection with the diagnostic test elements herein, an exemplary electrically conductive path comprises two WEs, a contact pad for each WE, and a respective WE conductive trace portion < RTI ID = 0.0 > . Likewise, the electrically conductive path includes a CE conductive trace portion for CE, CE contact pads, and CE electrically extended coupling to the contact pads. As used herein, " working electrode " or " WE " refers to an electrode to which an analyte is electrooxidized or electrochemically reduced with or without the aid of a mediator, while " And passes through it an electrochemical current having the same magnitude and opposite sign as the current passing through WE. The CE also includes an opposing electrode that also functions as a reference electrode ( i.e. , an opposing / reference electrode).

The electrode system may also include at least one sample sufficiency electrode (SSE), a sample sufficiency contact pad, and a respective conductive trace portion that extends between the SSE and SSE contact pads and electrically couples them. When included, SSEs can be used to implement various techniques for determining the sufficiency of bodily fluid samples applied to test elements. See, for example, International Patent Application Publication Nos. WO 2014/140170 and WO 2015/187580.

The electrode system is also disclosed in International Patent Application Publication Nos. (IE) that can be used to verify that the electrode system has not been damaged, as described in WO 2015/187580.

The electrode system is also disclosed in International Patent Application Publication Nos. WO < RTI ID = 0.0 > 2013/017218 < / RTI > May include information circuitry in the form of a plurality of selectable resistive elements forming a resistive network as described in U.S. Pat. No. 2015/0362455. The information encoded in the resistance network may be associated with attributes of the test elements, including, but not limited to, calibration information, test element type, manufacturing information, and the like.

Figure 2a illustrates an exemplary multiple analyte electrode system configuration for a diagnostic test element. In Figure 2a, the sample chamber of the test element comprises an electrode system of an electrically conductive material comprising a pair of SSEs positioned along each side edge of the nonconductive substrate, a first analyte located adjacent one of the SSEs Wherein the WE for measuring the second analyte has an electrode system of an electrically conductive material comprising a CE / WE pair for measuring a second analyte, WE for measuring a second analyte disposed adjacent to another SSE, And has a working area larger than that of WE.

Figure 2B illustrates another exemplary multi-analyte electrode system configuration for a diagnostic test element. In Figure 2B, the sample chamber of the test element comprises a pair of SSEs positioned along each side edge of the non-conductive substrate, a CE / WE pair for measuring the first analyte, and a WE for measuring the second analyte And an electrode system of an electrically conductive material. In contrast to FIG. 2A, the CE of FIG. 2B extends across the sample chamber in front of both WEs. Further, WE has the same working area.

Figure 2C illustrates another exemplary multi-analyte electrode system configuration for a diagnostic test element. In contrast to the configuration shown in FIG. 2-FIG. 2B, FIG. 2C includes WE for a second analyte that does not include SSE and has a working area larger than WE for the first analyte.

Detecting reagents which can be applied to the electrode system are described in detail above.

The above-mentioned detection reagent can be formulated as a viscous solution containing a thickener and a thixotropic agent to improve its physical properties. The thickener is selected to provide a thick liquid matrix with the residual components being homogeneously dispersed. Thickening agents and thixotropic agents also inhibit liquid or semi-paste material from flowing or spreading over the surface of the substrate after each being deposited and dried. After the detection reagent is deposited, it is quickly dried with a hydratable reagent matrix.

As such, the drying detection reagent may be first provided by dissolving these components in a solvent or solvent mixture, and then removing the solvent or solvent mixture by suitable treatment, as described further below.

As used herein, " dry " means that the reagent composition is essentially free of a solvent or solvent mixture. As used herein, "essentially free" means that at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93% of the solvent or solvent mixture originally present in the solution of the reagent composition , At least about 94%, at least about 95%, at least about 96%, at least about 97%, or even at least about 98% is removed from the composition. Accordingly, the solvent or solvent mixture may contain up to about 15%, up to about 10%, up to about 9% up to about 8% up to about 7% up to about 6% up to about 5% 3%, or up to about 2% by weight of the dry reagent composition. The percent value and other percentage values referred to herein refer to weight percent (w / w).

For example, a detection reagent may be applied to each electrode (s) via ink jetting. See, for example , U.S. Pat. 9,157,109. Alternatively, the detection reagent may be applied through a custom print drop for each respective electrode (s). Alternatively, the detection reagent can be applied via a PicoJet (R) dispensing system (Nordson EFD) to deposit the detection reagent (s) in a separate region of the same chamber. Other contact or non-contact dispensing systems described in the following paragraphs may also be used.

Alternatively, detection reagents may be applied to each electrode (s) via a vacuum assisted slot die coating. A method of controlling the thickness and uniformity of the detection reagent by vacuum assisted slot die coating is disclosed, for example, in International Patent Publication No. Hei. WO < RTI ID = 0.0 > 2012/139767 & 7,749,437.

Additional details regarding exemplary diagnostic test element configurations that may be used herein may be found in, for example, International Patent Application Publication Nos. WO 2014/037372, 2014/068022 and 2014/068024; United States Patent Application Publication Nos. 2003/0031592 and 2006/0003397; United States Patent Nos. 5,694,932; 5,271,895; 5,762,770; 5,948,695; 5,975,153; 5,997,817; 6,001,239; 6,025,203; 6,162,639; 6,207,000; 6,245,215; 6,271,045; 6,319,719; 6,406,672; 6,413, 395; 6,428,664; 6,447,657; 6,451,264; 6,455,324; 6,488,828; 6,506,575; 6,540,890; 6,562,210; 6,582,573; 6,592,815; 6,627,057; 6,638,772; 6,755,949; 6,767,440; 6,780,296; 6,780,651; 6,814,843; 6,814,844; 6,858,433; 6,866,758; 7,008,799; 7,025,836; 7,063,774; 7,067,320; 7,238,534; 7,473,398; 7,476,827; 7,479,211; 7,510,643; 7,727,467; 7,780,827; 7,820,451; 7,867,369; 7,892,849; 8,180,423; 8,298,401; 8,329,026, and RE42560, RE42924 and RE42953.

Likewise, the diagnostic test elements may include one or more reflective layers of one or more pigments having reflective properties, such as white pigments, such as, for example, titanium dioxide particles. In some cases, at least one of the reflective layers may be on the surface of the substrate facing away from the detection reagent, which may be in the form of a test field, thus functioning as a sample application surface. In this way, detection of at least one analyte can occur through the substrate from the side opposite the sample application surface. To facilitate this design, the substrate may be coated with at least one detection light that is completely or partially optically transparent to at least one excitation light illuminated with the detection reagent and / or reflected and / or emitted by the detection reagent , Where transparency is understood as a transparency of at least about 70%. In other cases, a liquid sample may be introduced laterally ( i.e. , in parallel with the layer structure) to the detection reagents.

A concern in multiple analyte diagnostic test elements is to control the potential crosstalk of the signals that may occur between or among the various detection reagents. Various methods of attenuating or avoiding crosstalk are known in the art and can be used in conjunction with the multiple analyte test elements disclosed herein. For example, (1) by using a detection reagent formulation that expands but is not completely soluble (e.g., the matrix material described above); (2) separating the detection reagents from each other; (3) by using a physical barrier between detection reagents ( i.e. , leaving residual conductive material or other materials; laser marking on the substrate); And / or (4) the use of a short time frame to complete the measurement so that reagent diffusion is limited.

Analytical measuring devices, devices and test systems

The test system may include an analyte measurement device and at least one multi-analyte diagnostic test element as described above.

FIG. 3 illustrates an exemplary analyte measurement system including an analyte measurement device, such as a test meter 38, operatively coupled to an electrochemical diagnostic test element 10. In particular, the test element is a multi-analyte diagnostic test element as described above.

Typically, the meter 38 and the diagnostic test element 10 are operable to determine the concentration of a plurality of analytes in the body fluid sample provided to the test element 10. In some cases, the sample may be a bodily fluid sample, such as whole blood, plasma, serum, urine, or saliva. In other instances, the sample may be another type of fluid sample that is tested for the presence or concentration of one or more electrochemically reactive analyte (s), such as aqueous environmental samples.

In Figure 3, the diagnostic test element 10 is a disposable test strip that is removably inserted into a connection terminal (or test element port) 40 of the meter 38. In some cases, the test element 10 comprises a dual analyte-glucose and ketone-test element and includes features and functions for electrochemically measuring glucose and ketone. In other instances, the test element 10 can be used to electrochemically analyze other analytes such as, for example, amino acids, antibodies, bacteria, carbohydrates, drugs, lipids, markers, nucleic acids, peptides, proteins, .

The meter 38 generally includes an entry (or input) means 44, a controller, a memory associated with the controller / microcontroller, and a programmable processor associated with and associated with the controller. The instrument also includes an output, such as electronic display 42, which is connected to the processor and used to display various types of information to the user, including analyte concentration (s) or other test results. Moreover, the meter 38 may include an associated test signal generation and measurement circuit (not shown) operable to generate a test signal, apply a signal to the test element 10, and measure one or more responses of the test element 10 to the test signal (Not shown). The processor is also coupled to a test element port and is operative to process and record data in memory in connection with detecting the presence and / or concentration of an analyte obtained through the use of a multi-analyte test element as described herein It is possible. The test element port includes a connector configured to engage a contact pad of the electrical system. The instrument also includes a user entry means coupled to the processor, which is accessible by a user to provide input to the processor, and wherein the processor also receives an input command from the user entry means and provides an output responsive to the input command It is programmable.

In addition, the processor may be coupled to a communication module or link to facilitate wireless transmission to the meter 38. In one form, the communication link includes a meter 38 and other devices or parties such as a social worker, a caregiver, a caregiver, a nurse, a pharmacist, a nurse including a primary care physician or a secondary care physician to provide just a few possibilities. , Or may be used to exchange messages, alerts, or other information between health care providers and emergency medical professionals. The communication link may also be used to download a programming update to the meter 38. By way of example and not limitation, the communication link may be implemented using mobile phone standard technology including third generation (3G) and fourth generation (4G) technologies, or via Bluetooth®, Zigbee®, Wibree, ultra-wide band (UWB), a wireless local area network (WLAN), a general packet radio service (GPRS), a WiMAX or a Worldwide Interoperability for Microwave Access (WiMAN), a Wireless Medical Telemetry (WMTS) Wireless Universal Serial Bus), Global System for Mobile communications (GSM), Short Message Service (SMS), or WLAN 802.11x standards.

Thus, the controller may comprise a hybrid combination of one or more components, state logic machines or other types of dedicated hardware, or programmable hardware and dedicated hardware, which may be configured and programmed in a single unit or multiple component form. One or more components of the controller may have electronic diversity that defines digital circuitry, analog circuitry, or both. As an addition to or as an alternative to electronic circuitry, the controller may include one or more mechanical or optical control elements.

In some cases involving electronic circuitry, the controller includes an integrated processor operatively coupled to one or more solid state memory devices that at least partially define the memory. In this manner, the memory includes operational logic executed by a processor that is a microprocessor and is arranged to read and write data to and from the memory in accordance with one or more routines of the program executed by the microprocessor.

The memory may also include one or more types of solid state electronic memory and may additionally or alternatively include magnetic or optical variety. For example, the memory may be a solid state electronic random access memory (RAM), a sequential access memory (SAM) (e.g., FIFO diversity or LIFO variety), a programmable read only memory (EPROM) An erasable programmable read only memory (EEPROM); Or a combination of these types. The memory may also be a hybrid combination of volatile, non-volatile, or volatile and non-volatile variety. Some or all of the memory may be of a portable type such as a disk, tape, memory stick, cartridge, code chip, and the like. The memory may be at least partially integrated with the processor and / or may be in the form of one or more components or units.

In some cases, the meter 38 is pluggable to a socket or other receiving means and is removable, in communication with a memory or controller, to provide information about calibration codes, measurement methods, measurement techniques, and information management A memory key can be used. Examples of such detachable memory keys are described, for example, in U.S. Pat. 5,366,609 and 5,053,199.

The controller may also be used to provide signal conditioners, filters, limiters, analog-to-digital (A / D) converters, digital-to-analog (D / A) converters, communication ports, Lt; / RTI >

Returning to the entry means 44, the entry means 44 may be used to input one or more other types of input devices, such as a keyboard, a mouse or other pointing device, a touch screen, a touch screen, a touch pad, a roller ball, The entry means 44 may be defined by a push-button input device.

Likewise, the display 42 may include one or more display devices such as a cathode ray tube (CRT) type, a liquid crystal display (LCD) type, a plasma type, an organic light emitting diode And output means. Other input and display means such as loudspeakers, voice generators, voice and speech recognition systems, haptic displays, electronic wired or wireless communication subsystems, and the like may be included.

As described above, the test element port 40 includes connectors configured to engage contact pads of the electrode system of the test elements described herein. The connection between the meter 38 and the diagnostic test element 10 is accomplished by applying a test signal having a potential or a series of potentials across the electrodes of the electrode system and subsequently generating by the detection reagents in the presence of an analyte of interest And is used to receive an electrochemical signal that can be correlated with the concentration of the analyte. In this manner, the processor is configured to evaluate an electrochemical signal to evaluate the presence and / or concentration of the analyte, and the results of the analyte can be stored in memory.

In some cases, the meter 38 can be configured as a blood glucose meter and includes the features and functions of the ACCU-CHEK® AVIVA® instrument as described in the booklet "Accu-Chek® Aviva Blood Glucose Meter Owner's Booklet" (2007) Part of which is incorporated herein by reference in its entirety. 6,645,368. In other instances, the meter 38 may be used to electrochemically measure other analytes such as, for example, amino acids, antibodies, bacteria, carbohydrates, drugs, lipids, markers, nucleic acids, proteins, peptides, . Additional details regarding exemplary instruments configured for use with electrochemical measurement methods are described, for example, in U.S. Pat. 4,720,372; 4,963,814; 4,999,582; 4,999,632; 5,243,516; 5,282,950; 5,366,609; 5,371,687; 5,379,214; 5,405,511; 5,438,271; 5,594,906; 6,134,504; 6,144,922; 6,413,213; 6,425,863; 6,635,167; 6,645,368; 6,787,109; 6,927,749; 6,945,955; 7,208,119; 7,291,107; 7,347,973; 7,569,126; 7,601,299; 7,638,095 and 8,431,408.

In addition to the instruments, the test systems include one or more multi-analyte diagnostic test elements as detailed above.

Another component that may be included in the test systems includes an incision device for obtaining body fluid samples. Examples of lancing devices include, for example, those described in International Patent Application Publication Nos. WO2002 / 089523 and WO2002 / 089524. In some cases, the lancing device may be integrated into the instrument. For example , see id .

Multi-analyte measurement method

The measurement methods disclosed herein primarily use current measurement; However, it is contemplated that the methods may be used in conjunction with other electrochemical measurement techniques ( e.g., coulometry, potentiometry, or voltammetry). In addition, the measurement methods can be implemented using advanced microprocessor-based algorithms and processes that result in dramatically improved system performance. These measurement methods also provide flexibility and various methods for generating algorithms that can achieve performance to be improved, such as 10/10 performance. As used herein, "10/10 performance" means that the measured blood glucose concentration is within about ± 10% of the actual blood glucose concentration for glucose concentrations> 100 mg / dl, and the glucose concentrations <100 mg / dl &lt; / RTI &gt; of the actual blood glucose concentration.

The measurement methods may include the steps described herein and these steps may, but need not, be performed in the sequence as described. However, other sequences can be conceived. Moreover, individual or multiple steps may be performed in parallel and / or overlapping in time and / or individually or in multiple repeated steps. In addition, these methods may include additional, non-specific steps.

In general, the methods of measurement begin with obtaining a bodily fluid sample suspected of having or possessing one or more analytes of interest. Examples of body fluids include, but are not limited to, blood, interstitial fluid, saliva, tears, and urine. As used herein, " blood " means whole blood and its cell-free components, plasma and serum.

When constructing the multi-analyte diagnostic test elements to test glucose and ketones, the body fluid sample is obtained by dissecting the fingertip or an approved alternative site ( e.g., forearm, palm, ear, brachium, calf and thigh) Fresh capillary can. In addition, a body fluid sample containing the analyte (s) of interest can be obtained and delivered to the test elements in any manner. Therefore, measurement methods are mainly concerned with the in vitro method. For example, a blood sample can be obtained in a conventional manner by incising the skin such as a lancet, needle or scalpel, and then contacting the test element with a blood sample that appears on the surface of the skin. Alternatively, the test elements may be used in conjunction with control fluids used in conventional manner to verify the integrity of the test system.

Generally, the diagnostic test elements are operable to evaluate a target analyte while using only a very small volume of a body fluid sample. In this way, only a small amount of skin incision is needed to produce the volume of body fluid required for the test, and pain and other concerns due to such a method can be minimized or eliminated.

After applying a body fluid sample to the dosing end of the diagnostic test element and re-hydrating the detection reagent, the method comprises applying an electrical test sequence to the electrode system of the test element. Such a test sequence may be supplied by a meter from its connection terminal to one or more contact pads of the electrode system.

Generally, the electrical test sequence comprises one or more AC blocks (optional) and / or one or more DC blocks, as is known in the art. See, for example, International Patent Application Publication Nos. WO 2014/140718, WO 2014/140164, WO 2010/140170, WO 2010/140172, WO 2011/140173, WO 2011/012270 and WO 2011/140177.

If included, measures the AC block of low amplitude signals associated with the DC block and the current response thereto. Figures 4A-4F illustrate exemplary test sequences that may be used in conjunction with SMBG and other test systems. As shown in FIGS. 4A and 4B, the test sequence may include one or more blocks of AC and / or DC potentials, which are described in more detail below.

As to the AC block, a plurality of AC segments, for example, about 2 segments to about 10 segments, about 3 segments to about 9 segments, about 4 segments to about 8 segments, about 5 segments To about 7 segments, or about 6 segments, and so on. In other examples, the AC block may include about two segments, about three segments, about four segments, about five segments, about six segments, about seven segments, about eight segments, about nine segments, Lt; / RTI &gt; In another example, the AC block may have more than 10 segments, i.e., about 15 segments, about 20 segments, or about 25 segments. In another example, an AC block may include one segment, wherein multiple low frequency AC signals are applied to the segment at the same time.

Those skilled in the art understand that the number of AC segments will be limited by the complexity of the response, the frequency range involved and the time available to perform measurements. Higher frequencies typically require higher bandwidth electronics and faster sampling, while lower frequencies take longer and are typically more noisy. The maximum number of segments will therefore be a compromise of these parameters, choosing the minimum coefficients and frequency ranges needed to distinguish between sample and environmental and / or confusing interest factors.

The frequency of each signal within each segment of the AC block may range from about 1 kHz to about 20 kHz, from about 2 kHz to about 19 kHz, from about 3 kHz to about 18 kHz, from about 4 kHz to about 17 kHz, From about 6 kHz to about 15 kHz, from about 7 kHz to about 14 kHz, from about 8 kHz to about 13 kHz, from about 9 kHz to about 12 kHz, or from about 10 kHz to about 11 kHz. In other examples, the frequency of each segment in the AC block is about 1 kHz, about 2 kHz, about 3 kHz, about 4 kHz, about 5 kHz, about 6 kHz, about 7 kHz, about 8 kHz, about 9 kHz, about 10 about 11 kHz, about 13 kHz, about 14 kHz, about 15 kHz, about 16 kHz, about 17 kHz, about 18 kHz, about 19 kHz, or about 20 kHz. In another example, the frequency of each signal in each segment of the AC block may be greater than 20 kHz, i.e., about 30 kHz, about 40 kHz, or about 50 kHz. In some cases, one or more segments may have the same frequency, while in other cases, each segment has a different frequency than the other segments. However, four frequencies are generally appropriate. The exact frequencies adopted can be easily generated by simple constants of the maximum frequency of the measuring system clock.

However, the maximum frequency limitation of a signal in one segment of the AC block is possible up to 100 kHz for battery-powered portable devices such as meters. If it is exceeded, increasing demands on analog bandwidth, sampling rate, storage and processing speed are added quickly, while the imaginary portion of conventional biosensor response becomes smaller with frequency. The lower the frequency, the longer it takes and the longer it takes to sample with comparable accuracy.

The AC block typically includes at least two different low-amplitude signals. For example, the AC block may include two (2) segments at two (2) frequencies, such as about 10 kHz or about 20 kHz followed by about 1 kHz or about 2 kHz. In other cases, the AC block includes a plurality of low-amplitude signals. For example, the AC block may have five (5) segments at four (4) frequencies, such as about 10 kHz, about 20 kHz, about 10 kHz, about 2 kHz, and about 1 kHz. Alternatively, the AC block may have four (4) segments at four (4) frequencies, such as about 20 kHz, about 10 kHz, about 2 kHz, and about 1 kHz. Alternately, the AC block may have four (4) frequencies simultaneously applied at about 10 kHz, about 20 kHz, about 10 kHz, about 2 kHz, and about 1 kHz. Alternatively, the AC block may have a multi-frequency excitation waveform that simultaneously applies the desired low-amplitude AC signals. The AC frequencies may be sequentially applied, concurrently coupled and applied, or may be analyzed through a Fourier transform.

The block of low-amplitude AC signals may be applied for between about 500 msec and about 1.5 sec, between about 600 msec and about 1.25 sec, between about 700 msec and about 1000 msec, or between about 800 msec and about 900 msec. Alternatively, the block of low-amplitude AC signals may be applied for about 500 msec, about 600 msec, about 700 msec, about 800 msec, about 900 msec, about 1000 msec, about 1.25 seconds, or about 1.5 seconds. In particular, the block of low-amplitude AC signals may be applied for about 100 msec to about 300 msec.

However, those skilled in the art will appreciate that the number, frequency, duration, and sequence of AC segments may vary.

AC current response information can be obtained at any time during the test sequence. Impedance results at lower frequencies can be affected by analyte concentration if the electrochemical cell is obtained after DC polarization. In some cases, a series of AC current response measurements may be obtained early in the test sequence. Measurements taken immediately after the fluid sample is applied to the test element will be affected by diffusion, temperature and reagent solubility. In other cases, the AC-responsive current measurements may be obtained at a sufficient time after the appropriate sample is applied to allow the response to stabilize and avoid the initial transient response. Likewise, the response current measurements can be made at one or more frequencies. Due to their capacitive nature, multiple AC measurements spaced by a frequency octave or decade may provide different sensitivities or easier manipulation.

The response information for this AC block can be used to evaluate diffusion, temperature and reagent solubility before initiating analyte measurements. As a result, the response information to the AC block can be used to correct confusion parameters such as Hct and / or temperature, or to determine the suitability of the test element to provide the condition and correct result.

Additional details regarding exemplary AC blocks in electrochemical measurement methods are described, for example, in U.S. Pat. 7,338,639; 7,390,667; 7,407,811; 7,417,811; 7,452,457; 7,488,601; 7,494,816; 7,597,793; 7,638,033; 7,751,864; 7,977,112; 7,981,363; 8,148,164; 8,298,828; 8,377,707 and 8,420,404.

With respect to one exemplary DC block for a multi-analyte test sequence, it may include, for example, from about 2 pulses to about 10 pulses, from about 3 pulses to about 9 pulses, from about 4 pulses About 8 pulses, about 5 pulses to about 7 pulses, or about 6 pulses. In other cases, the DC block may comprise about two pulses, about three pulses, about four pulses, about five pulses, about six pulses, about seven pulses, about eight pulses, about nine pulses, About 10 pulses may be included. In other cases, the DC block may have more than 10 pulses, i.e., about 15 pulses, about 20 pulses, or about 25 pulses. As used herein, "pulse" means at least one excitation and one recovery period.

The DC block typically comprises a constantly applied potential difference alternating between about 0 mV and about +450 mV potential difference, or a slow time-varying potential difference that can be analyzed by conventional DC electrochemical methods. However, one of ordinary skill in the art understands that the range for applied potential differences will vary and will vary depending on the analyte and the reagent chemistry used. As such, the excitation pulse potential may be greater than, less than, or equal to about +450 mV. Examples of excitons include, but are not limited to, 50 mV, 75 mV, 100 mV, 125 mV, 150 mV, 175 mV, 200 mV, 225 mV, 250 mV, 275 mV, 300 mV, 325 mV, mV, 400 mV, 425 mV, 450 mV, 475 mV, 500 mV, 525 mV, 550 mV, 575 mV, 600 mV, 625 mV, 650 mV, 675 mV, 700 mV, 725 mV, 750 mV, 775 mV, 800 mV, 825 mV, 850 mV, 875 mV, 900 mV, 925 mV, 950 mV, 975 mV or 1000 mV.

Regardless of the number of DC pulses, each DC pulse is between about 50 msec and about 500 msec, between about 60 msec and about 450 msec, between about 70 msec and about 400 msec, between about 80 msec and about 350 msec, between about 90 msec and about 300 msec From about 100 msec to about 250 msec, from about 150 msec to about 200 msec, or about 175 msec. Alternatively, each pulse may be about 50 msec, about 60 msec, about 70 msec, about 80 msec, about 90 msec, about 100 msec, about 125 msec, about 150 msec, about 175 msec, about 200 msec, about 225 about 300 msec, about 250 msec, about 250 msec, about 275 msec, about 300 msec, about 325 msec, about 350 msec, about 375 msec, about 400 msec, about 425 msec, about 450 msec, about 475 msec, . In particular, each DC pulse at +450 mV can be applied for about 250 msec, and each DC pulse at 0 mV can be applied for about 500 msec. Alternatively, each pulse may be applied for less than about 50 msec or greater than about 500 msec.

Generally, the ramp rate of each DC pulse is selected to provide a reduction of about 50% or more in peak current relative to the peak current provided by the nearly ideal potential transition. In some cases, each pulse may have the same ramp rate. In other cases, some pulses may have the same ramp rate and other pulses may have different ramp rates. In other cases, each pulse has its own ramp rate. For example, the effective ramp rate may be from about 5 mV / msec to about 75 mV / msec, or from about 10 mV / msec to about 50 mV / msec, from 15 mV / msec to about 25 mV / msec, Lt; / RTI &gt; Alternatively, the ramp rate may be about 5 mV / msec, about 10 mV / msec, about 15 mV / msec, about 20 mV / msec, about 25 mV / msec, about 30 mV / msec, about 35 mV / About 40 mV / msec, about 45 mV / msec, about 50 mV / msec, about 55 mV / msec, about 60 mV / msec, about 65 mV / msec, about 70 mV / msec or about 75 mV / msec. In particular, the ramp rate may be from about 40 mV / msec to about 50 mV / msec.

In the DC block, the applied DC potential can be fixed at approximately 0 mV between pulses to provide a recovery pulse, which generally produces a continuous excitation waveform. This is in contrast to test sequences generally known in the art that preclude the possibility of collecting and analyzing the current between positive pulses, thereby defining the use of an open circuit between positive DC pulses. As used herein, a " recovery pulse " means that the electrochemical reaction with an analyte of interest ( e.g. , glucose) is converted to " off " so that, prior to subsequent irradiation with a different amount of DC pulse Means a zero-potential pulse ( e.g. , about -10 mV to about +10 mV) applied during a sufficiently long recovery period, causing the system to return to a fixed starting point.

Thus, the exemplary DC block can alternate between about 0 mV and about +450 mV (in dual current measurement mode).

The response information for such a DC block may be used to assess the presence or presence of a first analyte such as glucose concentration or presence. Additionally, information such as the recovery current response, shape and / or size from the DC block potential can be used to correct the Hct and / or temperature as well as the wetting of the reagent and sample diffusion, and the thickness variation of the detection reagent.

As with the AC block, those skilled in the art understand that the number, potential, duration, and sequence of DC pulses can vary.

With respect to another exemplary DC block for a multi-analyte test sequence, it may include, for example, from about 2 intervals to about 10 intervals, from about 3 intervals to about 9 intervals, from about 4 intervals About 8 intervals, about 5 intervals to about 7 intervals, or about 6 intervals. In other instances, the waveform may include about 1 interval, about 2 intervals, about 3 intervals, about 4 intervals, about 5 intervals, about 6 intervals, about 7 intervals, about 8 intervals, about 9 intervals Or about 10 intervals. In other cases, the waveform may have intervals greater than 10, i.e., about 15 intervals, about 20 intervals, or about 25 intervals. However, the number of waveform intervals is typically limited to the time available for the test sequence.

The waveform intervals may be alternating between alternating positive or negative potentials and vice versa. For example, the potential may be about -450 mV to about +450 mV, about -425 mV to about +425 mV, about -400 mV to about +400 mV, about -375 mV to about +375 mV, about -350 mV About -275 mV to about +250 mV, about -232 mV to about +325 mV, about -300 mV to about +300 mV, about -275 mV to about +275 mV, about -250 mV to about +250 mV, About -125 mV to about +150 mV, about -100 mV to about +150 mV, about -125 mV to about +150 mV, about -100 mV to about +150 mV, about -200 mV to about +200 mV, About +100 mV, about -75 mV to about +75 mV, or about -50 mv to about +50 mV. In some cases, one or more of the consecutive cycles may have the same potential, while in other cases the consecutive cycles have discrete dislocations from other segments.

Regardless of that number, each of the waveform intervals may be from about 100 msec to about 5 sec, from about 200 msec to about 4 sec, from about 300 msec to about 3 sec, from about 400 msec to about 2 sec, from about 500 msec to about 1 sec , About 600 msec to about 900 msec, or about 700 msec to about 800 msec. Alternatively, each waveform interval may be about 100 milliseconds, about 150 milliseconds, about 200 milliseconds, about 250 milliseconds, about 300 milliseconds, about 350 milliseconds, about 400 milliseconds, about 450 milliseconds, about 500 milliseconds, About 600 msec, about 700 msec, about 750 msec, about 800 msec, about 850 msec, about 900 msec, about 950 msec, about 1 sec, about 1.5 sec, about 2 sec, about 2.5 sec, about 3 sec , About 3.5 sec, about 4 sec, about 4.5 sec, or about 5 sec. In particular, each waveform interval at about -450 mV can be applied for about 100 msec to about 200 msec, and each waveform interval at about +450 mV can be applied for about 100 msec to about 200 msec. Alternatively, each waveform interval may be applied for less than about 100 msec or greater than about 5 msec.

In some cases, the waveform intervals may have the same ramp rate. In other cases, some waveform intervals may have the same ramp rate, and other waveform intervals may have different ramp rates. In other cases, each waveform interval has its own ramp rate. For example, the ramp rate may be about 0.5 mV / msec to 45 mV / msec. Alternatively, the ramp rate of each interval may be from about 1 mV / msec to about 40 mV / msec, from about 2 mV / msec to about 30 mV / msec, from about 3 mV / msec to about 20 mV / about 6 mV / msec to about 17 mV / msec, about 7 mV / msec to about 16 mV / msec, about 8 mV / msec, To about 15 mV / msec, from about 9 mV / msec to about 14 mV / msec, or from about 10 mV / msec to about 13 mV / msec, or from about 11 mV / msec to about 12 mV / msec. Alternatively, the ramp rate of each interval may be from about 0.5 mV / msec to about 2 mV / msec, from about 3 mV / msec to about 4 mV / msec, from about 5 mV / msec to about 6 mV / msec to about 8 mV / msec, from about 9 mV / msec to about 10 mV / msec, from about 11 mV / msec to about 12 mV / msec, from about 14 mV / msec to about 16 mV / Msec to about 20 mV / msec, or from about 30 mV / msec to about 35 mV / msec, or from about 40 mV / msec to about 45 mV / msec. In particular, the ramp rate is between about 3 mV / msec and about 9 mV / msec, such as about 5.1 mV / msec or about 7.15 mV / msec.

In some cases, the waveform may be a triangular waveform, a trapezoidal waveform, a sinusoidal waveform, or combinations thereof.

Such a DC block can be used to assess the concentration or presence of a second analyte such as ketone concentration or presence. In addition, information such as recovery current response, shape and / or size from DC block potentials can also be used in combination with antioxidants ( e.g., ascorbate, citric acid, diferoxamine (DFO), glutathione, N-acetylcysteine ), Pyrrolidine dithiocarbamate (PDTC), triradid-mesylate (TLM), and uric acid).

As such, those skilled in the art will appreciate that the number, potential, duration, and sequence of DC pulses can be varied.

With respect to another exemplary DC block for a multi-analyte test sequence, it may include, for example, from about 2 intervals to about 10 intervals, from about 3 intervals to about 9 intervals, from about 4 intervals About 8 intervals, about 5 intervals to about 7 intervals, or about 6 intervals. In other instances, the waveform may include about 1 interval, about 2 intervals, about 3 intervals, about 4 intervals, about 5 intervals, about 6 intervals, about 7 intervals, about 8 intervals, about 9 intervals Or about 10 intervals. In other cases, the waveform may have intervals greater than 10, i.e., about 15 intervals, about 20 intervals, or about 25 intervals. However, the number of waveform intervals is typically limited to the time available for the test sequence.

The waveform intervals may be alternating between alternating positive or negative potentials and vice versa. For example, the potential can range from about 0 mV to about +250 mV, from about 0 mV to about +225 mV, from about 0 mV to about +200 mV, from about 0 mV to about +175 mV, at about 0 mV To about +150 mV, from about 0 mV to about +125 mV, from about -0 mV to about +100 mV. In other instances, the potential can be maintained at about 250 mV, about 225 mV, about 200 mV, about 175 mV, about 150 mV, about 125 mV, or about 100 mV. In some cases, one or more of the consecutive cycles may have the same potential, while in other cases the consecutive cycles have discrete dislocations from other segments.

Regardless of the number, each waveform interval can range from about 100 msec to about 5 sec, from about 200 msec to about 4 sec, from about 300 msec to about 3 sec, from about 400 msec to about 2 sec, from about 500 msec To about 1 sec, from about 600 msec to about 900 msec, or from about 700 msec to about 800 msec. Alternatively, each waveform interval may be about 100 milliseconds, about 150 milliseconds, about 200 milliseconds, about 250 milliseconds, about 300 milliseconds, about 350 milliseconds, about 400 milliseconds, about 450 milliseconds, about 500 milliseconds, About 600 msec, about 700 msec, about 750 msec, about 800 msec, about 850 msec, about 900 msec, about 950 msec, about 1 sec, about 1.5 sec, about 2 sec, about 2.5 sec, about 3 sec , About 3.5 sec, about 4 sec, about 4.5 sec, or about 5 sec. In particular, each waveform interval at about -450 mV can be applied for about 100 msec to about 200 msec, and each waveform interval at about +450 mV can be applied for about 100 msec to about 200 msec. Alternatively, each waveform interval may be applied for less than about 100 msec or greater than about 5 msec.

In some cases, the waveform intervals may have the same ramp rate. In other cases, some waveform intervals may have the same ramp rate, and other waveform intervals may have different ramp rates. In other cases, each waveform interval has its own ramp rate. For example, the ramp rate may be about 0.5 mV / msec to 45 mV / msec. Alternatively, the ramp rate of each interval may be from about 1 mV / msec to about 40 mV / msec, from about 2 mV / msec to about 30 mV / msec, from about 3 mV / msec to about 20 mV / about 6 mV / msec to about 17 mV / msec, about 7 mV / msec to about 16 mV / msec, about 8 mV / msec, To about 15 mV / msec, from about 9 mV / msec to about 14 mV / msec, or from about 10 mV / msec to about 13 mV / msec, or from about 11 mV / msec to about 12 mV / msec. Alternatively, the ramp rate of each interval may be about 5 mV / msec, about 1 mV / msec, about 2 mV / msec, about 3 mV / msec, about 4 mV / msec, about 7 mV / msec, about 8 mV / msec, about 9 mV / msec, about 10 mV / msec, about 11 mV / msec, About 15 mV / msec, about 16 mV / msec, about 17 mV / msec, about 18 mV / msec, about 19 mV / msec, about 20 mV / msec, about 25 mV / mV / msec, about 40 mV / msec, or about 45 mV / msec. In particular, the ramp rate is between about 3 mV / msec and about 9 mV / msec, such as about 5.1 mV / msec or about 7.15 mV / msec.

In some cases, the waveform may be a triangular waveform, a trapezoidal waveform, a sinusoidal waveform, or combinations thereof.

The response information for such a DC block can be used to assess the concentration or presence of the first analyte, such as ketone concentration or presence.

As such, those skilled in the art will appreciate that the number, potential, duration, and sequence of DC pulses can be varied.

An exemplary multi-analyte test sequence is shown in Figure 4a (left panel), which includes (1) a first fixed DC potential difference between a first pair of electrodes dedicated to a first analyte measurement, and (2) And a second fixed DC potential difference between a second pair of electrodes dedicated to analyte measurement. Advantageously, only one potentiometer is required by sequentially measuring the analyte. Sequence order can be determined by analytes that benefit from longer reaction times. Thus, the first analyte is measured dynamically while the WE of the second analyte is maintained as an open circuit. Sequentially, WE of the first analyte is an open circuit while WE of the second analyte is connected to a potentiometer. The potentials applied in (1) and (2) may vary depending on the mediator. The selectable potentiometer gain can be advantageous if the physiological level is significantly different. 4A (right panel) shows an exemplary response to the test sequence shown in FIG. 4A (left panel).

An alternate exemplary multi-analyte test sequence is shown in Figure 4b (left panel), which is a delay that allows (1) a sample to be introduced into the test element to allow the reaction to proceed, while the open circuit or near- (2) a first fixed DC potential difference sufficient to generate a Faraday current between the first pair of electrodes for first analytical measurement only, and (3) a first analyte And a first fixed DC potential difference sufficient to generate a Faraday current between the first pair of electrodes for measurement only. Figure 4b (right panel) shows an exemplary response to the test sequence shown in Figure 4b (left panel).

An alternate exemplary multi-analyte test sequence is shown in Figure 4c (left panel), with (1) a delay introduced by the sample into the test element to allow the reaction to proceed, while the open circuit or near- (2) a first fixed DC potential difference sufficient to generate a Faraday current between the first pair of electrodes dedicated to the first analyte measurement, and (3) the current returned to zero (4) a second fixed DC potential difference sufficient to generate a Faraday current between the second pair of electrodes for the second analyte measurement only. Figure 4c (right panel) shows an exemplary response to the test sequence shown in Figure 4c (left panel).

An alternative exemplary multi-analyte test sequence is shown in FIG. 4d (left panel), which illustrates (1) a delay in which a sample is introduced into a test element to allow the reaction to proceed, while an open circuit or near- (2) a short duration of about +450 mV pulses separated by a similar short duration (e.g., about 50-500 msec) recovery intervals to which about 0 mV potential difference is applied A fixed potential difference of about +175 mV followed by an open circuit between the first DC block of duration (e.g., about 50-500 msec), followed by (3) a second electrode pair dedicated to the second analyte measurement And a second DC block to which the second DC block is applied. Figure 4d (right panel) shows an exemplary response to the test sequence shown in Figure 4d (left panel).

An alternative exemplary multi-analyte test sequence is shown in Figure 4e (left panel), which includes not only the DC components discussed above, but also one or more AC components. For example, the test sequence may include (1) a delay after the sample is introduced into the test element to allow the reaction to proceed, while the open circuit or near zero volt potential difference is maintained between both electrode pairs, (2) AC blocks of amplitude AC signals, and (3) about 0 mV recovery potential over a 10 msec interval, separated by a similarly short duration (e.g., about 50-500 msec) A first DC block of pulses of short duration (e.g., about 50-500 msec) ramped to 450 mV, (4) a pulse having alternating or circulating pulses at about -450 mV to about +450 mV in the closed circuit 2 DC block, and (5) a third DC block that applies a fixed potential difference of about +175 mV following the open circuit between the second pair of electrodes dedicated to the second analyte measurement. Figure 4e (right panel) shows an exemplary response to the test sequence shown in Figure 4e (left panel).

In a multiple analyte test sequence, the AC component may be a series of small amplitude excitations at multiple discrete frequencies. Likewise, the first (pulsed) DC component is applied across the pair of primary electrodes between an amplitude sufficient to produce a Faraday current response proportional to the concentration of the primary analyte ( i.e. , +450 mV in this example) A series of slew rate control potential differences. The amplitude of the potential depends on the mediator of the primary analyte. The duration of the positive amount of pulse is long enough to minimize the effect of the charging current and is ideally less than the thickness of the hydrated detection reagent at 10-15 μm (d = √ (D _M × t) Lt; 150 msec to limit the diffusion distance on the primary analyte WE. It is advisable to measure the current response of the primary analyte at the end of one or more positive pulses to minimize the effects of overcharging and other noises. Pulses with positive 130 msec pulses are scattered at 0 V applied potential difference intervals of sufficient duration to allow the electrochemical cell to approximate its initial state (I → 0).

Moreover, the second DC component emulates a cyclic voltammetric technique. Here, the potential difference applied to the pair of primary electrodes is swept between +450 mV and -450 mV at a rate of 3.5 V / sec. The second DC component may be used to detect an electroactive interferer. Followed by a third DC component for measuring the secondary analyte concentration.

Similar to the first DC component, the third DC component depends on the secondary analyte mediator and has an amplitude that is sufficient to produce a Faraday current response proportional to the concentration of the secondary analyte ( i . E., +175 mV in this example) to 2 One or more slew rate controlled potential difference (s) is applied across the pair of the secondary electrodes. The current of the secondary analyte is measured once after application of the secondary potential, and is typically about 500 msec.

An alternative exemplary multi-analyte test sequence including the AC and DC components as well as the burn-off intervals as described above is shown in FIG. 4f. For example, the test sequence may be (1) a first positive potential difference applied between one or both electrode pairs for a short period of time, resulting in a reduction of the concentration of the reduced mediator present in the reagent (2) an AC block of a plurality of low amplitude AC signals; (3) ramped over a 10 msec interval with a second amount of potential difference sufficient to generate a measurable Faraday current associated with the first analyte concentration from about 0 V, and then back again to about 0 V, Similarly, for a short duration, the potential recovery circuit is closed (e.g., about 50-500 msec) separated by a recovery pulse, short duration (e.g., about 50-500 msec) of claim 1 DC pulse of (4) a second DC component having pulses alternating or circulating between about -450 mV and about +450 mV in the closed circuit, and (5) a second DC component having pulses alternating or circulating between about -450 mV and about +450 mV in the closed circuit, Is ramped over a 10 msec interval with a third amount of potential difference sufficient to generate the current, then ramped back to about 0 V, and a similarly short duration ( e.g., , About 50-500 msec) Recovery pulse By a separate, short duration (e.g., about 50-500 msec) includes a first 3 DC component of the pulse. Figure 4f (right panel) shows an exemplary response to the test sequence shown in Figure 4f (left panel).

With this in mind, one of the test signals described herein may be applied to one or more WEs to provide a potential difference between WE and CE. Alternatively, a test potential other than virtual ground or reference potential may be provided as CE to provide a potential difference between WE and CE. It should be appreciated that a variety of other additional and alternative test cells, electrodes and / or circuit configurations as described above may be used that are operative to contact the combined sample and detection reagent to apply a test signal to the electrode system and measure the response thereto do.

The AC and / or DC current response information is collected in the applied test sequence and includes the current response to the AC and DC blocks. Important information includes, but is not limited to, the duration, shape and / or size of the current response to the excitation pulse and / or the recovery pulse of the test sequence. In some cases, the current-response information can be collected at the A / D sampling rate for DC and AC measurements to simplify the system design by including a single shared signal path for AC and DC measurements. Typical digital audio sampling rate ranges include, but are not limited to, about 44.1 kHz to about 192 kHz. A / D converters in this range are readily available from a variety of commercial semiconductor suppliers.

The current response information for the AC block can be used to determine impedance, admittance, and phase values or other complex parameters as described in more detail below. Likewise, the information of the current to the DC block may be used to determine the concentration of the analyte or other complex parameters ( e.g. , Hct-, temperature- and / or interferometer-based compensation and / or calibration, Reagent film thickness and reaction kinetics and / or calibration).

In the method, the AC and / or DC response current information may be from about 2,000 / sec to about 200,000 / sec, from about 3,000 / sec to about 190,000 / sec, from about 4,000 / sec to about 180,000 / sec, Sec, from about 6,000 / sec to about 160,000 / sec, from about 7,000 / sec to about 150,000 / sec, from about 8,000 / sec to about 140,000 / sec, from about 9,000 / sec to about 130,000 / sec, from about 10,000 / sec to about 120,000 sec About 20,000 / sec to about 100,000 / sec, about 30,000 / sec to about 90,000 / sec, about 40,000 / sec to about 80,000 / sec, about 50,000 / sec to about 70,000 / sec , Or about 60,000 / sec (i.e., measured or recorded). In some examples, the AC and / or DC response current information is from about 100 / sec to about 200 / sec, from about 200 / sec to about 300 / sec, from about 300 / sec to about 400 / sec, sec, from about 500 / sec to about 600 / sec, from about 600 / sec to about 700 / sec, from about 700 / sec to about 800 / sec, from about 800 / sec to about 900 / sec, about 2,000 / sec to about 2,500 / sec, about 2,500 / sec to about 3,000 / sec, about 3,000 / sec to about 3,500 / sec, about 3,500 / sec to about 4,000 sec, from about 4,000 / sec to about 4,500 / sec, from about 4,500 / sec to about 5,000 / sec, from about 5,000 / sec to about 5,500 / sec, from about 5,500 / sec to about 6,000 / sec, from about 6,000 / sec to about 6,500 sec, from about 6,500 to about 7,000 / sec, from about 7,000 / sec to about 7,500 / sec, from about 7,500 / sec to about 8,000 / sec, from about 8,000 / sec to about 8,500 / sec, from about 8,500 to about 9,000 / Sec to about 20,000 / sec, from about 9,000 / sec to about 9,500 / sec, from about 9,500 / sec to about 10,000 / sec, from about 10,000 / sec to about 30,000 / sec, from about 30,000 / sec to about 40,000 / sec, from about 40,000 / sec to about 50,000 / sec, from about 50,000 / sec to about 60,000 / sec, from about 60,000 / sec to about 70,000 / sec, sec to about 80,000 / sec, from about 80,000 / sec to about 90,000 / sec, from about 90,000 / sec to about 100,000 / sec, from about 100,000 / sec to about 110,000 / sec, from about 110,000 / sec to about 120,000 / sec, sec to about 130,000 / sec, from about 130,000 / sec to about 140,000 / sec, from about 140,000 / sec to about 150,000 / sec, from about 150,000 / sec to about 160,000 / sec, from about 160,000 / sec to about 170,000 / sec, sec to about 180,000 / sec, from about 180,000 / sec to about 190,000 / sec, or from about 200,000 / sec. In other instances, the AC and / or DC response current information may be up to about 100 / sec, about 200 / sec, about 300 / sec, about 400 / sec, about 500 / sec, sec, about 1,200 / sec, about 1,500 / sec, about 1,750 / sec, about 2,000 / sec, about 2,225 / sec, about 2,500 / sec, about 2,750 / sec, about 3,000 sec, about 3,250 / sec, about 3,500 / sec, about 3,750 / sec, about 4,000 / sec, about 4,250 / sec, about 4,500 / sec, about 4,750 / sec, about 5,000 / sec, about 5,250 / sec, about 5,750 / sec, about 6,000 / sec, about 6,250 / sec, about 6,500, about 7,000 / sec, about 7,250 / sec, about 7,500 / sec, about 7,750 / sec, about 8,000 / sec, about 8,250 / sec About 9,500 / sec, about 9,750 / sec, about 10,000 / sec, about 15,000 / sec, about 20,000 / sec, about 25,000 / sec, about 8,500 / sec, about 8,500 / sec, about 8,750, about 9,000 / sec, about 9,250 / About 60,000 / sec, about 65,000 / sec, about 70,000 / sec, about 75,000 / sec, about 30,000 / sec, about 35,000 / sec, about 40,000 / sec, about 45,000 / sec, about 50,000 / sec, about 55,000 / About 80,000 / sec, about 85,000 / sec, about 90,000 / sec, about 95,000 / sec, about 100,000 / sec sec, about 125,000 / sec, about 130,000 / sec, about 135,000 / sec, about 140,000 / sec, about 145,000 / sec, about 150,000 / sec, sec, about 155,000 / sec, about 160,000 / sec, about 165,000 / sec, about 170,000 / sec, about 175,000 / sec, about 180,000 / sec, about 185,000 / sec, about 190,000 / sec, about 195,000, &Lt; / RTI &gt; In yet another example, AC and / or DC response current information may be obtained above 200,000 / sec.

Additional details regarding exemplary electrochemical measurement methods are described, for example, in U.S. Pat. 4,008,448; 4,225,410; 4,233,029; 4,323,536; 4,891,319; 4,919,770; 4,963,814; 4,999,582; 4,999,632; 5,053,199; 5,108,564; 5,120,420; 5,122,244; 5,128,015; 5,243,516; 5,288,636; 5,352,351; 5,366,609; 5,385,846; 5,405,511; 5,413,690; 5,437,999; 5,438,271; 5,508,171; 5,526,111; 5,627,075; 5,628,890; 5,682,884; 5,727,548; 5,762,770; 5,858,691; 5,997,817; 6,004,441; 6,054,039; 6254736; 6,270,637; 6,645,368; 6,662,439; 7,073,246; 7,018,843; 7,018,848; 7,045,054; 7,115,362; 7,276,146; 7,276,147; 7,335,286; 7,338,639; 7,386,937; 7,390,667; 7,407,811; 7,429,865; 7,452,457; 7,488,601; 7,494,816; 7,545,148; 7,556,723; 7,569,126; 7,597,793; 7,638,033; 7,731,835; 7,751,864; 7,977,112; 7,981,363; 8,148,164; 8,298,828; 8,329,026; 8,377,707; And 8,420,404, and RE36268, RE42560, RE42924 and RE42953. Other exemplary electrochemical measurement methods that may be used herein are described in International Patent Application Publication Nos. WO 2014/140718; WO 2014/140164; WO 2014/140170; WO 2014/140172; WO 2014/140173; And WO 2014/140177.

The analyte concentration may be determined by an algorithm and / or correlation of the amount of redox equivalent ( e. G., Electrons) measured and measured by the electrode system with such algorithm and / or correlation known in the art , Lt; / RTI &gt;

After the response information has been processed and correlated to determine the analyte concentration, the method may include displaying to the user one or more analyte concentrations or trends on the instrument. Various graphics and / or numerical means for displaying data and other related information to a user are known in the art. For example, U.S. Patent Application Laid-Open Publication No. Hei. 2009/0210249 and U.S. Pat. 9,218,453 .

In addition to the steps described above, these methods may include additional steps. In connection with the determination of glucose and ketone, the method comprises determining both analytes for each test, and determining one analyte ( e.g. , glucose), another analyte ( e.g. , a ketone) And providing a glucose concentration to the user if a predetermined threshold or condition for all of the analytes is not met. For example, a hydroxybutyrate concentration of less than 0.6 mM is regarded as normal, while a concentration of hydroxybutyrate between 0.6 mM and 1.5 mM may be problematic and a risk of developing DKA above 1.5 mM. Concentrations of hydroxybutyrate in excess of 3 mM in blood are indicative or DKA and require emergency treatment. Thus, in some cases, the glucose concentration is displayed to the user and the ketone concentration is displayed only when the predetermined threshold (s) or condition (s) are met, and the predetermined threshold (s) or condition A glucose concentration of 240 mg / dL or a ketone concentration of about 0.6 mM to about 3.0 mM, or even about 0.6 mM to about 1.5 mM. For example, in International Patent Application Laid-Open Publication No. Hei. 2014/068024.

In other instances, the method also includes providing an indication in response to determining that the first analyte concentration exceeds a predetermined value, the method comprising the steps of displaying a first analyte concentration, providing an alert, Providing a list of actions to be taken in response to the first analyte concentration exceeding a predetermined value; and transmitting a message to at least one of a user, a health care provider, a caregiver, and a parent or guardian of the test element .

Specifically, providing an indication in response to determining whether the first analyte concentration exceeds a predetermined level may comprise sending a message to the mobile device or computer. In some cases, providing an indication in response to determining that the first analyte concentration exceeds a predetermined level further comprises displaying a message associated with the first analyte concentration on a test meter. In other cases, providing an indication in response to determining that the first analyte concentration exceeds a predetermined level includes displaying a message associated with the first analyte concentration. In other cases, providing an indication in response to determining that the first analyte concentration exceeds a predetermined level includes changing the color or shade of at least a portion of the display screen or text display. In another aspect, providing an indication in response to determining that the first analyte concentration exceeds a predetermined level comprises displaying an information icon on a display screen. In another aspect, providing an indication in response to determining that the first analyte concentration exceeds a predetermined level includes displaying an information icon on the display screen with audio tones or vibrations to induce the patient to pay attention do. In one aspect of this form, the method further comprises providing a message in response to the selection of the information icon. In another aspect, the message includes at least one of a description of the first analyte concentration, an action list to be taken in response to the first analyte concentration exceeding a predetermined level, and contact information of the healthcare provider.

Thus, the ketone clock can be set by the instrument whenever a measured glucose value greater than or equal to a predetermined value, such as 240 mg / dL, is recorded. Alternatively, the ketone clock can be set by the meter whenever the measured ketone value is above a predetermined value, e.g., 0.6 mM to 3.0 mM or more. The ketone clock recommends testing glucose and ketones every 4-6 hours as long as the predetermined value is maintained. In one non-limiting manner, for example, during the initiation and on the way of a ketone watch, the instrument can automatically display the measured glucose and ketone levels regardless of their relationship to the pre-specified values. The ketone clock may start a new set of trend data to determine if the ketone has started to rise even though the ketone is below the threshold of the higher ketone level. If the user indicates that there is a disease such as a cold or flu, a kettle clock may also be started.

Examples

The concept of the present invention will be more fully understood in view of the following non-limiting embodiments which are provided for the purpose of illustration and not of limitation.

Example 1: Ketone and glucose detection reagents for dual analyte analysis.

Methods: Ketone and glucose detection reagents were prepared as described below. Table 3 shows the basic components of the ketone detection reagent.

Table 4 shows the basic components of the glucose detection reagent.

3-HB was prepared in a 150 mM phosphate buffer, pH 7.

The test sequence was applied to different levels of 3-HB in the buffer ( i.e. , 0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0 and 8.0 mM). International patent application publication no. A test sequence as described in WO 2014/140178 was used, followed by a single long pulse of 175 mV (for the glucose counter electrode). For ketone measurements, the current was read at 0.5 second after applying a 175 mV potential difference between the ketone working electrode and the glucose counter electrode.

The ketone reagent had a high medium content (10 mM) as compared to other studies of the present application. The polymer content of the dried film was higher than the other studies.

Results: Figure 5 shows the linear response during a dose response study in which an increasing current as the 3-HB concentration increases is measured.

Example 2: Aqueous crosstalk study.

Methods: Ketone and glucose detection reagents were prepared as in Example 1 above. The detection reagent was incorporated into the test element and then used in the crosstalk experiment. The glucose detection reagent was applied to the glucose working electrode and the counter electrode, and the ketone detecting reagent was applied to the ketone working electrode.

The aqueous matrix for glucose and 3-HB was 150 mM phosphate buffer, pH 7.

In the crosstalk experiment, aqueous samples containing various concentrations of 3-HB and glucose (0, 1.0, 3.0 and 8.0 mM for 3-HB; and 0 or 300 mg / dL for glucose) were administered to the test elements . At about 130 msec after initiating a first ramp pulse of between about 0 mV and about +450 mV between the glucose working electrode for glucose measurement and the glucose counter electrode ( e.g. , Figure 4e, point "DC1" in the right panel) ), And the current was read at about 0.5 second after applying a 175 mV potential difference between the ketone working electrode and the glucose counter electrode for ketone measurement.

Results: Figure 6a shows the glucose current in the presence of 3-HB and does not affect the glucose current due to the presence of different levels of 3-HB (0 mM, 1 mM, 3 mM and 8 mM). Two glucose levels were tested, each with a different level of 3-Hb.

Similarly, Figure 6b shows the 3-HB current in the presence of glucose and does not affect the 3-HB current due to the presence of different levels of glucose (0 mg / dL and 300 mg / dL). Four different levels of 3-HB (0 mM, 1 mM, 3 mM and 8 mM) were tested and each level contained 0 mg / dL or 300 mg / dL of glucose.

Example 3: Effect of different coenzyme on ketone detection reagent.

Methods: Reagent chemicals were prepared as described in Example 1 above. Here, however, for the coenzyme / cofactor, ketone detection reagents were prepared using NAD instead of cNAD. Tables 5 and 6 show the basic components of an alternative ketone detection reagent. In the administration reaction test, the glucose reagent is the same as the above example.

Test sequence was like in the above examples, different levels of 3-HB in the buffer (that is, 0, 0.5, 1.0, 1.5, 2.0, 3.0 and 4.0 mM), and glucose in the different levels of the buffer (that is, 0 , 57, 123, 520 and 1000 mg / dL). As in Example 2 above, at about 130 msec ( for example , at the point " DC1 &quot;) after initiating the first ramp pulse from about 0 mV to about +450 mV between the glucose working electrode for glucose measurement and the glucose counter electrode "), And the current was read at about 0.5 s after applying a 175 mV potential difference between the ketone working electrode and the glucose counter electrode for ketone measurement.

The ketone reagent has a low mediator content (7.5 mM) as compared to the above studies. Likewise, the polymer content in the dried film is lower than the above-mentioned study.

Results: Figure 7a shows that NAD (diamond) is slightly more effective as a cofactor than cNAD (square); The response to cNAD is still acceptable, in particular because cNAD has improved stability over natural co-factor NAD.

Figure 7b shows that there is a slight difference in glucose measurement when NAD or cNAD is used in the ketone detection reagent. Thus, Figure 7b shows that the glucose response is not interrupted by the detection reagent deposition method ( e . G., PicoJet®).

Example 4: Effect of different mediators on ketone detection reagents.

Methods: Reagent chemicals were prepared as described in Example 1 above. However, here, the ketone detection reagent was prepared with cPES instead of PG355. Table 7 shows the basic components of the ketone detection reagent. In the administration reaction study, the glucose reagent is the same as the above example.

The test sequence was the same as in the previous example and was applied to various levels of 3-HB in the buffer ( i.e. , 0, 0.5, 1.0, 1.5, 2.0, 3.0 and 4.0 mM). As described above, the current was read at 0.5 second after applying a 175 mV potential difference between the ketone working electrode and the glucose counter electrode for ketone measurements.

The ketone reagent has a low medium content (7.5 mM) when compared to the above studies. Likewise, the polymer content in the dried film is lower than the above-mentioned study.

Results: Figure 8 shows that cPES is an effective mediator with comparable results using PG355.

Example 5: Effect of different enzymes on ketone detection reagents.

Methods: Reagent chemicals were prepared as described in Example 2 above. Here, however, ketone detection reagents were prepared with wild-type HBDH, AFDH3 HBDH, and AFDH4 HBDH mutants (see EP Patent Application No. 16165421.5 for additional details on mutants). Table 8 shows the basic components of the alternative ketone detection reagent. In the administration reaction study, the glucose detection reagent was the same as in Examples 1 and 2 above.

The test sequence was the same as in the previous example and different levels of 3-HB ( i.e. , 0.5, 1.5, and 3.0 mM) and different levels of glucose in the buffer ( i.e. , 40, 150, and 400 mg / dL). The sample was also prepared as glycosized venous blood with a hematocrit of about 41%. As in the above Example 2-3, and then in sayi glucose working electrode for measuring glucose and glucose counter electrodes initiating a first ramp pulse of from about 0 mV to about +450 mV in about 130 msec (for example, point &Quot; DC1 &quot;), and the current was read at about 0.5 second after applying a 175 mV potential difference between the ketone working electrode and the glucose counter electrode for ketone measurement.

Results: Overall, among the different HBDH enzymes, no significant effect on the 3-HB signal ( i.e. , current) was observed in the presence of glucose ( see FIGS. 9a-9c). Likewise, no significant effect on glucose signal (see FIG. 9d-9f) was observed in the presence of 3-HB. Therefore, there is no evidence of crosstalk between reagents.

Example 6: Alternative ketone and glucose detection reagents for dual analyte analysis.

Methods: Reagent chemicals were prepared as described in Example 2 above. However, the glucose detection reagent was prepared using FAD-GDH and NA1144, and the ketone detection reagent was prepared using HBDH and diaphorase / NA1144. The concentration of NA1144 for the glucose detection reagent was 25 mM and 7.5 mM for the ketone detection reagent.

The ketone and glucose detection reagents were deposited using PicoJet® diacid dispensing.

The test sequence was the same as in the previous example and the samples with different levels of 3-HB ( i.e. 0 mM, 1 mM, 3 mM and 8 mM) in the buffer and a single glucose level ( i.e. 300 mm / dL) .

Results: As shown in Figure 10a, when the test solution contained different levels of 3-HB (0, 0.5, 1.5, 4 and 8 mM) and the strip was administered either from the front or from the side, Glucose concentration = 300 mg / dL) was not observed. Likewise, there was no significant effect on the 3-HB signal ( i.e., current) when the test strip was administered from either the front or side of the strip, as shown in Figure 10b. When the strip was administered side-by-side, the test solution first flowed on top of the glucose reagent. If the glucose reagent is not firmly held in place and crosstalk occurs between the reagents, it is expected that there will be some effect on the 3HB current measured at the ketone working electrode.

Example 7: Slot die coating of double glucose detection reagent on test element.

Methods: Reagent chemicals were prepared as described in Example 1 above. Likewise, the test sequence is the same as that described in the first embodiment.

The ketone and glucose detection reagents were deposited using a dual reagent slot die coating instead of PicoJet® discrete dispensing.

For the crosstalk experiment, the test elements contained different levels of 3-HB ( i.e. , 0.5, 1.5, and 3.0 mM) and different levels of glucose in the buffer ( i.e. , 1, 150, and 300 mg / Was administered. Also, as in Example 2 above, at about 130 msec after initiating the first ramp pulse of between about 0 mV and about +450 mV between the glucose working electrode for glucose measurement and the glucose counter electrode ( e.g., &Quot; DC1 &quot;), and the current was read at about 0.5 second after applying a 175 mV potential difference between the ketone working electrode and the glucose counter electrode for ketone measurement.

Results: Overall, no significant effect on the 3-HB signal was observed when different levels of glucose were present ( see FIG. 11A). Similarly, no significant effect on the glucose signal was observed in the presence of 3-HB (see FIG. 11B). Thus, there was no evidence of crosstalk between reagents through slot die coating.

Example 8: Ink-jet printing double detection reagent with polymer overcoat on top of test element.

Methods: Reagent chemicals were prepared as described in Example 1 above. Here, one glucose detection reagent contained FAD-GDH and the other glucose detection reagent was a mixture of two glucose, including mutant PQQ-GDH (Roche Diagnostics, Inc., Indianapolis, USA) with low maltose sensitivity A detection reagent was prepared. The ink jet formulations for each glucose detection reagent were the same except for the enzyme and coenzyme.

Two glucose detection reagents were deposited using inkjet printing instead of dual reagent slot die coating or PicoJet® discrete dispensing.

The test sequence is the same as that described in the first embodiment. The minimum crosstalk between the electrodes was observed for the major perturbations for each electrode. Because the WE region is different for each reagent, the current response is normalized to the glucose response for each electrode. No dose response data was collected; Instead, the experiment was to run a "kinetic" experiment with 450 mV across each WE and CE, where the current was monitored from the time the sample was applied.

Results: Figure 12a shows the reaction of 350 mg / dL glucose, 350 mg / dL maltose or 350 mg / dL xylose on the electrode with low maltose sensitivity mutant PQQ-GDH. No significant xylose response was observed on the electrode with the mutant PQQ-GDH. When crosstalk occurs due to the FAD-GDH reagent, a considerable xylose response is expected to occur.

Figure 12B shows the reaction of 350 mg / dL glucose, 350 mg / dL maltose and 350 mg / dL xylose on the FAD-GDH electrode. A minimal maltose reaction was observed on the FAD-GDH electrode. Some signal due to xylose was observed, which was expected due to xylose interference with FAD-GDH.

All of the patents, patent applications, patent application disclosures and other publications mentioned in this specification are incorporated herein by reference as if their entirety were developed.

The concepts of the present invention have been described in connection with what is presently considered to be the most practical and preferred embodiments. However, the concept of the present invention is presented in an illustrative manner and is not intended to be limited to the disclosed embodiments. Accordingly, it will be appreciated by those of ordinary skill in the art that the concept of the present invention is intended to cover all modifications and alternative arrangements within the spirit and scope of the inventive concept as evolved from the appended claims. The numbered embodiments are presented below.

Numbered embodiments

In addition to or as an alternative to the above, the following embodiments are described:

One. As the drying detection reagent,

A first detection reagent comprising a first enzyme-dependent enzyme or a substrate for a first enzyme, a first coenzyme, and a first medium;

And a second detection reagent comprising a second enzyme-dependent enzyme or a substrate for a second enzyme, a second coenzyme, and a second medium, wherein the second coenzyme-dependent enzyme, the second coenzyme, Wherein the second agent has a different type and / or concentration as compared to the first reagent.

2. Wherein the first coenzyme dependent enzyme and the second coenzyme dependent enzyme are selected from the group consisting of an alcohol dehydrogenase, a glucose dehydrogenase, a glucose-6-phosphate dehydrogenase, a glucose oxidase, a glycerol dehydrogenase, a hydroxybutyrate dehydrogenase, a malate dehydrogenase, (FAD), nicotinamide adenine dinucleotide (NAD) - or pyrroloquinoline-quinone (PQQ) -dependent oxidase or dehydrogenase And the enzyme is selected from the group consisting of enzymes.

3. The dry detection reagent according to Embodiment 1 or 2, wherein the first coenzyme-dependent enzyme is a glucose dehydrogenase, a glucose-6-phosphate dehydrogenase, or a glucose oxidase.

4. The dry detection reagent according to any one of Embodiments 1 to 3, wherein the second coenzyme-dependent enzyme is a hydroxybutyrate dehydrogenase.

5. The dry detection reagent according to Embodiment 1, wherein the first coenzyme-dependent enzyme is a glucose dehydrogenase and the second coenzyme-dependent enzyme is a hydroxybutyrate dehydrogenase.

6. The dry detection reagent according to Embodiment 1, wherein both the first coenzyme-dependent enzyme and the second coenzyme-dependent enzyme are glucose dehydrogenase, glucose oxidase, or hydroxybutyrate dehydrogenase.

7. Wherein the first coenzyme and the second coenzyme are selected from the group consisting of flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), pyrroloquinoline-quinone (PQQ), thio-NAD, thio-NADP, PQQ, ) Or a salt thereof, or an artificial coenzyme such as a reduced form thereof, and the compound according to the above formula (I) is as follows:

Where:

A = adenine or an analogue thereof,

T = independently at each occurrence represents O or S,

U = independently for each occurrence represents OH, SH, BH ₃ ^- , or BCNH ₂ ^-

V = independently for each occurrence represents OH or phosphate group,

W is COOR, CON (R) ₂ , COR, or CSN (R) ₂ , wherein R in each case independently represents H or C ₁ -C ₂ -alkyl,

X _{1 and} X ₂ each independently represent O, CH ₂ , CHCH ₃ , C (CH ₃ ) ₂ , NH, or NCH ₃ ,

Y = NH, S, O, or CH ₂ ,

Z = optionally O, S and moieties comprising a cyclic having 5 C atoms including a heteroatom selected, and optionally one or more substituents from the N, and CR4 ₂ is during a residue CR4 ₂ coupled to a cyclic group and X ₂ ,

Where R4 = independently for each, is H, F, Cl or CH _3, but Z and pyridine moiety is not linked by a glycosidic bond,

&Lt; / RTI &gt; or a salt thereof or an optionally reduced form thereof.

8. The dry detection reagent according to any one of embodiments 1 to 7, wherein the first coenzyme is FAD, NAD, NADP, or a compound according to formula (I) or a salt thereof or a reduced form thereof.

9. The dry detection reagent according to any one of embodiments 1 to 7, wherein the first coenzyme is FAD.

10. The dry detection reagent according to any one of embodiments 1 to 9, wherein the second coenzyme is carbazole-NAD, carbazole-NADP, thio-NAD or thio-NADP.

11. The dry detection reagent according to any one of embodiments 1 to 7, wherein the first coenzyme is FAD and the second coenzyme is carbazole-NAD.

12. The dry detection reagent according to any one of embodiments 1 to 7, wherein both the first coenzyme and the second coenzyme are carbazole-NAD or PQQ.

13. Wherein the first and second intermediates are selected from the group consisting of azo compounds or azo precursors, benzoquinone, melphalan blue, nitrosoaniline or nitrosoaniline precursors, phenazine or phenazine precursors, quinone or quinone derivatives, thiazine or thiazine Wherein any of Embodiments 1 to 12 selected from the group consisting of a transition metal complex, a transition metal complex, such as a potassium ferricyanide and an osmium derivative, and a combination of a phenazine / phenazine precursor and a hexaamine ruthenium chloride, One dry detection reagent.

14. The dry detection reagent of any of embodiments 1 to 13, wherein said first agent is a nitrosoaniline derivative or a nitrosoaniline-based precursor, a ferricyanide, ruthenium hexamine, or phenazine.

15. The dry detection reagent according to any one of embodiments 1 to 13, wherein the first medium is N, N-bis (hydroxyethyl) -3-methoxy-4-nitrosoaniline hydrochloride.

16. The drying detection reagent according to any one of embodiments 1 to 13, wherein the second medium is a medolla blue, a phenazine or a phenazine precursor, or a quinone or a quinone derivative.

17. The drying detection reagent according to any one of embodiments 1 to 16, wherein the second medium is 1- (3-carboxy-propionylamino) -5-ethyl-phenazin-5-yumine.

18. Wherein the first agent is N, N-bis (hydroxyethyl) -3-methoxy-4-nitrosoaniline hydrochloride and the second agent is 1- (3-carboxy-propionylamino) &Lt; / RTI &gt; phenanthridin-5-ynyl-phenazin-5-ium.

19. The dry detection reagent according to any one of embodiments 1 to 12, wherein both the first medium and the second medium are N, N-bis (hydroxyethyl) -3-methoxy-4-nitrosoaniline hydrochloride.

20. Wherein the first coenzyme-dependent enzyme is an FAD-dependent glucose dehydrogenase, the first coenzyme is FAD and the first mediator is N, N-bis (hydroxyethyl) -3-methoxy-4-nitrosoaniline (NA1144), and the second coenzyme-dependent enzyme is a hydroxybutyrate dehydrogenase, and the second coenzyme is carba-NAD, carba-NADP, thio-NAD or thio-NADP, The dry detection reagent of embodiment 1, wherein the detection reagent is 1- (3-carboxy-propionylamino) -5-ethyl-phenazin-5-ium.

21. Wherein the first coenzyme-dependent enzyme is an FAD-dependent glucose dehydrogenase, the first coenzyme is FAD and the first mediator is N, N-bis (hydroxyethyl) -3-methoxy-4-nitrosoaniline Chloride, and the second coenzyme-dependent enzyme is an FAD-dependent glucose dehydrogenase or glucose oxidase, the second coenzyme is FAD or PQQ, and the second medium is a ferricyanide or nitrosoaniline other than NA1144 , The drying detection reagent of Embodiment 1.

22. The dry detection reagent according to any one of embodiments 1 to 21, wherein the first coenzyme-dependent enzyme and the first coenzyme are covalently bonded or ion-bonded to each other.

23. The dry detection reagent according to any one of embodiments 1 to 22, wherein the second coenzyme-dependent enzyme and the second coenzyme are covalently bonded or ion-bonded to each other.

24. As a diagnostic test element,

cover;

A nonconductive substrate comprising a capillary channel defined above, the nonconductive substrate formed to have a portion of the cover at a first end of the nonconductive substrate;

Wherein the first electrode system comprises a first counter electrode, a first working electrode, a first counter electrode lead, a first working electrode lead, a first counter electrode contact pad, and a second counter electrode pad on the nonconductive substrate, Wherein the first opposing electrode lead electrically connects the first opposing electrode to the first opposing electrode contact pad and the first working electrode lead connects the first working electrode to the first opposing electrode contact pad, The first electrode system electrically connected to the first working electrode contact pad and at least the first opposing electrode and the first working electrode are located in the region of the capillary channel;

A second electrode system provided on the non-conductive substrate at a position differentiated from a position of the first electrode system, the second electrode system comprising a second working electrode, a second working electrode lead, and a second working electrode contact pad Wherein the second working electrode lead electrically connects the second working electrode to the second working electrode contact pad and at least the second working electrode is located in the region of the capillary channel, system;

The dry detection reagent of any one of embodiments 1 to 23, wherein the first detection reagent is applied to the first electrode system and the second detection reagent is applied to the second electrode system, The dry detection reagent located in the region of the channel; And

And a spacer positioned between said cover and said nonconductive substrate, said spacer comprising an edge defining a boundary of said capillary channel.

25. 24. The diagnostic test element of embodiment 24, wherein the capillary channel comprises an inlet at a first end of the nonconductive substrate and an edge of the spacer extends between opposing side edges of the nonconductive substrate.

26. Wherein an edge of the cover extends across the first counter electrode lead, the first working electrode lead, and the second working electrode lead, and the first counter electrode, the first working electrode, The diagnostic test element of embodiment 24 or 25, wherein the diagnostic test element is positioned entirely within the capillary channel.

27. Further comprising at least two sample sufficiency electrodes disposed on the nonconductive substrate, wherein each sample sufficiency electrode of the sample sufficiency electrodes is located along a respective side edge of the nonconductive substrate. 24. A diagnostic test element according to any one of claims 24 to 26.

28. 27. The diagnostic test element of any one of embodiments 24-27, wherein the first working electrode has a working area equal to the working area of the second electrode.

29. 27. The diagnostic test element of any one of embodiments 24-27, wherein the first working electrode has a working area less than the working area of the second working electrode.

30. As a test system,

A test meter configured to analyze a body fluid sample; And

[0044] 24. A test system comprising one or more diagnostic test elements of any one of embodiments 24-29.

31. A method for electrochemically measuring the concentration or presence in a body fluid sample of one or more analytes of interest,

The bodily fluid sample suspected of having or having the one or more analytes of interest to hydrate the dry detection reagent in fluid contact with the bodily fluid sample is provided in any of embodiments 24-29 To a diagnostic test element of the system;

Applying an electrical test sequence to the diagnostic test element through a test meter configured to interact with the diagnostic test element,

a. A first fixed direct current (DC) component comprising a potential difference applied between the first counter electrode and the first working electrode to measure a first analyte of interest; And

b. A second fixed DC component including a potential difference applied between the first counter electrode and the second working electrode to measure a second analyte of interest,

Applying an electrical test sequence to the diagnostic test element;

Measuring response information for each component of the electrical test sequence with the test instrument; And

And determining the one or more analyte concentrations with the test instrument using the response information. &Lt; Desc / Clms Page number 20 &gt;

32. Wherein the electrical test sequence further comprises a delay after applying the body fluid sample to the diagnostic test element such that the body fluid sample is able to hydrate the dry detection reagent and the delay comprises a delay between the first counter electrode and the first counter electrode 31. The method of embodiment 31 comprising the step of: providing an open or near 0 V potential difference held between the working electrode and between the first opposing electrode and the second working electrode.

33. The electrical test sequence further comprises a near 0 V DC potential difference held between the first pair of electrodes such that a response current returns to zero between the first fixed DC component and the second fixed DC component , &Lt; / RTI &gt;

34. Wherein the first fixed DC component is a plurality of potential pulses ramped to about 0 V or about 0 V to about +450 mV, each pulse having a potential difference of about 0 mV between the first counter electrode and the first working electrode Wherein the second fixed DC component is about +175 mV potential difference applied between the first counter electrode and the second working electrode along a last recovery interval and the first fixed The method of embodiment 31 wherein the pulses and recovery intervals of the DC component are each between about 50 msec and about 500 msec and the second fixed DC component is at least about 500 msec.

35. Wherein the first fixed DC component is a plurality of potential pulses ramped to about 0 V or about 0 V to about +450 mV, each pulse having a potential difference of about 0 mV between the first counter electrode and the first working electrode And the second fixed DC component is a plurality of potential pulses ramped to about +175 mV at about 0 mV or about 0 mV along the last recovery interval and each pulse is separated by a recovery interval applied between each pulse Is separated by a recovery interval in which a potential difference of about 0 mV is applied between the first counter electrode and the second working electrode and the pulses and recovery intervals of the first fixed DC component and the second fixed DC component RTI ID = 0.0 &gt; 50 msec &lt; / RTI &gt; and about 500 msec, respectively.

36. 34. The method of embodiment 34 or 35 wherein the potential pulses of the first fixed DC component are ramped for about 10 msec.

37. Wherein the electrical test sequence further comprises a third fixed DC component and wherein the third fixed DC component comprises a plurality of potential pulses alternating between about -450 mV and about +450 mV, The method of embodiment 31, wherein a DC component is applied between the first fixed DC component and the second fixed DC component.

38. 31. The method as in any of the embodiments 31-37, wherein the electrical test sequence further comprises an alternating current (AC) component, wherein the AC component comprises a plurality of low amplitude AC signals.

39. 40. The method of embodiment 38, wherein said AC component comprises frequencies of about 10 kHz, about 20 kHz, about 10 kHz, about 2 kHz, and about 1 kHz, and each frequency is applied for about 0.5 seconds to about 1.5 seconds.

40. 40. The method of embodiment 38, wherein said AC component comprises frequencies of about 20 kHz, about 10 kHz, about 2 kHz, and about 1 kHz, and each frequency is applied for about 0.5 seconds to about 1.5 seconds.

41. 40. The method of embodiment 38, wherein the AC component is applied prior to the first fixed DC component and the second fixed component.

42. The electrical test sequence is characterized in that a positive potential difference is applied between the first opposing electrode and the first working electrode and alternatively between the first opposing electrode and the second working electrode to cause significant contribution prior to one or more analytes of interest Further comprising a burn-off interval to reduce the amount of reduced medium present in the detection reagent, wherein the burn-off interval is applied for about 0.5 seconds to about 1.0 seconds. Way.

43. 43. The method as in any of the embodiments 31-42, further comprising modulating treatment or modifying the diet based on the one or more analyte concentrations.

44. Further comprising sending a message to at least one of a user, a healthcare provider, a caregiver, and a parent or guardian of the test element to adjust treatment or modify the diet based on the one or more analyte concentrations. 31. The method according to any one of modes 31 to 43.

45. The method of embodiment 31, wherein said first analyte is glucose and said second analyte is hydroxybutyrate.

46. The method of embodiment 45, wherein said dry detection reagent is the dry detection reagent of embodiment 20.

47. The method of any one of embodiments 31-44, wherein the first analyte and the second analyte are the same.

48. [0215] 47. The method of embodiment 47, wherein said analyte is glucose.

49. 48. The method of embodiment 48, wherein said drying detection reagent is the drying detection reagent of embodiment 21.

50. A drying detection reagent substantially as described and illustrated herein.

51. A diagnostic test element substantially as described and illustrated herein.

52. A test system substantially as described and illustrated herein.

53. A method for electrochemically measuring the concentration or presence in a body fluid sample of one or more analytes of interest substantially as described and illustrated herein.

10 Diagnostic Test Elements
12 support substrate
14 Spacer
16 cover
18 First surface
20 second surface
22 First end
24 second end
26 side edge
28 side edge
30 capillary channel
32 end edge
34 internal surface
36 Lower surface
38 Instruments
40 Test element port
42 display
44 Entry means

Claims (49)

As the drying detection reagent,
A first detection reagent comprising a first enzyme-dependent enzyme or a substrate for a first enzyme, a first coenzyme, and a first medium;
A second coenzyme-dependent enzyme, a second coenzyme or a second coenzyme, and a second detecting reagent comprising a second coenzyme-dependent enzyme or a substrate for a second enzyme, a second coenzyme, and a second medium, Wherein at least one of the second medium has a different type and / or concentration as compared to the first detection reagent. The method according to claim 1,
Wherein the first coenzyme dependent enzyme and the second coenzyme dependent enzyme are selected from the group consisting of an alcohol dehydrogenase, a glucose dehydrogenase, a glucose-6-phosphate dehydrogenase, a glucose oxidase, a glycerol dehydrogenase, a hydroxybutyrate dehydrogenase, a malate dehydrogenase, (FAD), nicotinamide adenine dinucleotide (NAD) - or pyrroloquinoline-quinone (PQQ) -dependent oxidase or dehydrogenase &Lt; RTI ID = 0.0 &gt; enzymes. &Lt; / RTI &gt; 3. The method of claim 2,
Wherein the first coenzyme-dependent enzyme is a glucose dehydrogenase, a glucose-6-phosphate dehydrogenase, or a glucose oxidase. 3. The method of claim 2,
Wherein the second coenzyme-dependent enzyme is a hydroxybutyrate dehydrogenase. 3. The method of claim 2,
Wherein the first coenzyme-dependent enzyme is a glucose dehydrogenase and the second coenzyme-dependent enzyme is a hydroxybutyrate dehydrogenase. 3. The method of claim 2,
Wherein the first coenzyme-dependent enzyme and the second coenzyme-dependent enzyme are both glucose dehydrogenase, glucose oxidase, or hydroxybutyrate dehydrogenase. The method according to claim 1,
Wherein the first coenzyme and the second coenzyme are selected from the group consisting of flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), pyrroloquinoline-quinone (PQQ), thio-NAD, thio-NADP, PQQ, ) Or a salt thereof, or an artificial coenzyme such as a reduced form thereof, and the compound according to the above formula (I) is as follows.

Where:
A = adenine or an analogue thereof,
T = independently at each occurrence represents O or S,
U = independently for each occurrence represents OH, SH, BH ₃ ^- , or BCNH ₂ ^-
V = independently for each occurrence represents OH or phosphate group,
W is COOR, CON (R) ₂ , COR, or CSN (R) ₂ , wherein R in each case independently represents H or C ₁ -C ₂ -alkyl,
X _{1 and} X ₂ each independently represent O, CH ₂ , CHCH ₃ , C (CH ₃ ) ₂ , NH, or NCH ₃ ,
Y = NH, S, O, or CH ₂ ,
Z = optionally O, S and moieties comprising a cyclic having 5 C atoms including a heteroatom selected, and optionally one or more substituents from the N, and CR4 ₂ is during a residue CR4 ₂ coupled to a cyclic group and X ₂ ,
Where R4 = independently for each, is H, F, Cl or CH _3, but Z and pyridine moiety is not linked by a glycosidic bond,
Or a salt thereof or an optionally reduced form thereof. 8. The method of claim 7,
Wherein the first coenzyme is FAD, NAD, NADP, or a compound according to formula (I) or a salt thereof or a reduced form thereof. 8. The method of claim 7,
Wherein the first coenzyme is FAD. 8. The method of claim 7,
Wherein the second coenzyme is carba-NAD, carba-NADP, thio-NAD or thio-NADP. 8. The method of claim 7,
Wherein the first coenzyme is FAD and the second coenzyme is carbazole-NAD. 8. The method of claim 7,
Wherein the first coenzyme and the second coenzyme are both carba-NAD or PQQ. The method according to claim 1,
Wherein the first and second intermediates are selected from the group consisting of azo compounds or azo precursors, benzoquinone, melphalan blue, nitrosoaniline or nitrosoaniline precursors, phenazine or phenazine precursors, quinone or quinone derivatives, thiazine or thiazine Derivatives, transition metal complexes such as potassium ferricyanide and osmium derivatives, and combinations of phenazine / phenazine precursors and hexaamine ruthenium chloride, and derivatives thereof. 14. The method of claim 13,
Wherein the first agent is a nitrosoaniline derivative or a nitrosoaniline-based precursor, a ferricyanide, ruthenium hexamine, or phenazine. 15. The method of claim 14,
Wherein the first agent is N, N-bis (hydroxyethyl) -3-methoxy-4-nitrosoaniline hydrochloride. 14. The method of claim 13,
Wherein the second mediator is a medol blue, a phenazine or phenazine precursor, or a quinone or quinone derivative. 17. The method of claim 16,
Wherein the second agent is 1- (3-carboxy-propionylamino) -5-ethyl-phenazin-5-ium. The method according to claim 1,
Wherein the first agent is N, N-bis (hydroxyethyl) -3-methoxy-4-nitrosoaniline hydrochloride and the second agent is 1- (3-carboxy-propionylamino) Phenanthine-5-ium, dry detection reagent. The method according to claim 1,
Wherein the first agent and the second agent are both N, N-bis (hydroxyethyl) -3-methoxy-4-nitrosoaniline hydrochloride. The method according to claim 1,
Wherein the first coenzyme-dependent enzyme is an FAD-dependent glucose dehydrogenase, the first coenzyme is FAD and the first mediator is N, N-bis (hydroxyethyl) -3-methoxy-4-nitrosoaniline (NA1144), and the second coenzyme-dependent enzyme is a hydroxybutyrate dehydrogenase, and the second coenzyme is carba-NAD, carba-NADP, thio-NAD or thio-NADP, 1- (3-Carboxy-propionylamino) -5-ethyl-phenazin-5-ynum, dry detection reagent. The method according to claim 1,
Wherein the first coenzyme-dependent enzyme is an FAD-dependent glucose dehydrogenase, the first coenzyme is FAD and the first mediator is N, N-bis (hydroxyethyl) -3-methoxy-4-nitrosoaniline Chloride, and the second coenzyme-dependent enzyme is an FAD-dependent glucose dehydrogenase or glucose oxidase, the second coenzyme is FAD or PQQ, and the second medium is a ferricyanide or nitrosoaniline other than NA1144 , Drying detection reagent. The method according to claim 1,
Wherein the first coenzyme-dependent enzyme and the first coenzyme are covalently bonded or ion-bonded to each other. The method according to claim 1,
Wherein the second coenzyme-dependent enzyme and the second coenzyme are covalently bonded or ion-bonded to each other. As a diagnostic test element,
cover;
A nonconductive substrate comprising a capillary channel defined above, the nonconductive substrate formed to have a portion of the cover at a first end of the nonconductive substrate;
Wherein the first electrode system comprises a first counter electrode, a first working electrode, a first counter electrode lead, a first working electrode lead, a first counter electrode contact pad, and a second counter electrode pad on the nonconductive substrate, Wherein the first opposing electrode lead electrically connects the first opposing electrode to the first opposing electrode contact pad and the first working electrode lead connects the first working electrode to the first opposing electrode contact pad, The first electrode system electrically connected to the first working electrode contact pad and at least the first opposing electrode and the first working electrode are located in the region of the capillary channel;
A second electrode system provided on the non-conductive substrate at a position differentiated from a position of the first electrode system, the second electrode system comprising a second working electrode, a second working electrode lead, and a second working electrode contact pad Wherein the second working electrode lead electrically connects the second working electrode to the second working electrode contact pad and at least the second working electrode is located in the region of the capillary channel, system;
The dry sensing reagent of claim 1, wherein the first sensing reagent is applied to the first electrode system and the second sensing reagent is applied to the second electrode system, wherein the dry sensing reagent is located in the region of the capillary channel Said drying detection reagent; And
And a spacer positioned between said cover and said nonconductive substrate, said spacer comprising an edge defining a boundary of said capillary channel. 25. The method of claim 24,
Wherein the capillary channel comprises an inlet at a first end of the nonconductive substrate and an edge of the spacer extends between opposing side edges of the nonconductive substrate. 25. The method of claim 24,
Wherein an edge of the cover extends across the first counter electrode lead, the first working electrode lead, and the second working electrode lead, and the first counter electrode, the first working electrode, To be located entirely within the capillary channel. 25. The method of claim 24,
Further comprising at least two sample sufficiency electrodes disposed on the nonconductive substrate, wherein each sample sufficiency electrode of the sample sufficiency electrodes is located along a respective side edge of the nonconductive substrate, Element. 25. The method of claim 24,
Wherein the first working electrode has a working area equal to the working area of the second electrode. 25. The method of claim 24,
Wherein the first working electrode has a working area less than the working area of the second working electrode. As a test system,
A test meter configured to analyze a body fluid sample; And
25. A test system comprising one or more diagnostic test elements of claim 24. A method for electrochemically measuring the concentration or presence in a body fluid sample of one or more analytes of interest,
Applying the body fluid sample suspected of having or having the one or more analytes of interest to the diagnostic test element of claim 24 to hydrate the dry detection reagent in fluid contact with the body fluid sample with the dry detection reagent;
Applying an electrical test sequence to the diagnostic test element through a test meter configured to interact with the diagnostic test element,
a. A first fixed direct current (DC) component comprising a potential difference applied between the first counter electrode and the first working electrode to measure a first analyte of interest; And
b. A second fixed DC component including a potential difference applied between the first counter electrode and the second working electrode to measure a second analyte of interest,
Applying an electrical test sequence to the diagnostic test element;
Measuring response information for each component of the electrical test sequence with the test instrument; And
And determining the one or more analyte concentrations with the test instrument using the response information. &Lt; Desc / Clms Page number 20 &gt; 32. The method of claim 31,
Wherein the electrical test sequence further comprises a delay after applying the body fluid sample to the diagnostic test element such that the body fluid sample is able to hydrate the dry detection reagent and the delay comprises a delay between the first counter electrode and the first counter electrode The method comprising electrochemically measuring the concentration or presence of one or more analytes comprising an open or near 0 V potential difference held between working electrodes and between the first opposing electrode and the second working electrode. 32. The method of claim 31,
Wherein the electrical test sequence further comprises a near 0 V DC potential difference held between the first pair of electrodes between the first fixed DC component and the second fixed DC component such that the response current returns to zero. A method of electrochemically measuring the concentration or presence of one or more analytes. 32. The method of claim 31,
Wherein the first fixed DC component is a plurality of potential pulses ramped to about 0 V or about 0 V to about +450 mV, each pulse having a potential difference of about 0 mV between the first counter electrode and the first working electrode Wherein the second fixed DC component is about +175 mV potential difference applied between the first counter electrode and the second working electrode along a last recovery interval and the first fixed Wherein the pulses and the recovery intervals of the DC component are each between about 50 msec and about 500 msec and the second fixed DC component is at least about 500 msec. Way. 32. The method of claim 31,
Wherein the first fixed DC component is a plurality of potential pulses ramped to about 0 V or about 0 V to about +450 mV, each pulse having a potential difference of about 0 mV between the first counter electrode and the first working electrode And the second fixed DC component is a plurality of potential pulses ramped to about +175 mV at about 0 mV or about 0 mV along the last recovery interval and each pulse is separated by a recovery interval applied between each pulse Is separated by a recovery interval in which a potential difference of about 0 mV is applied between the first counter electrode and the second working electrode and the pulses and recovery intervals of the first fixed DC component and the second fixed DC component Wherein the concentration or presence of one or more analytes is between about 50 msec and about 500 msec, respectively. 35. The method according to claim 34 or 35,
Wherein the potential pulses of the first fixed DC component are ramped for about 10 milliseconds to electrochemically measure the concentration or presence of one or more analytes. 32. The method of claim 31,
Wherein the electrical test sequence further comprises a third fixed DC component and wherein the third fixed DC component comprises a plurality of potential pulses alternating between about -450 mV and about +450 mV, Wherein the DC component is applied between the first fixed DC component and the second fixed DC component. 32. The method of claim 31,
Wherein the electrical test sequence further comprises an alternating current (AC) component, the AC component comprising a plurality of low amplitude AC signals. 39. The method of claim 38,
Wherein the AC component comprises at least one of the following: an electrochemical concentration or presence of one or more analytes, wherein the AC component comprises frequencies of about 10 kHz, about 20 kHz, about 10 kHz, about 2 kHz, and about 1 kHz, . 39. The method of claim 38,
Wherein the AC component comprises an electrochemical measurement of the concentration or presence of one or more analytes, wherein the AC component comprises frequencies of about 20 kHz, about 10 kHz, about 2 kHz, and about 1 kHz, each frequency being applied for about 0.5 seconds to about 1.5 seconds Way. 39. The method of claim 38,
Wherein the AC component is applied prior to the first fixed DC component and the second fixed component. 32. The method of claim 31,
The electrical test sequence is characterized in that a positive potential difference is applied between the first opposing electrode and the first working electrode and alternatively between the first opposing electrode and the second working electrode to cause significant contribution prior to one or more analytes of interest Further comprising a burn-off interval to reduce the amount of reduced medium present in the detection reagent, wherein the burn-off interval is selected from the group consisting of a concentration or presence of one or more analytes A method for electrochemical measurement. 32. The method of claim 31,
Wherein the method further comprises adjusting the treatment or modifying the diet based on the one or more analyte concentrations. 32. The method of claim 31,
Further comprising sending a message to at least one of a user, a healthcare provider, a caregiver, and a parent or guardian of the test element to adjust therapy or modify the diet based on the one or more analyte concentrations. Wherein the concentration or presence of the above analytes is measured electrochemically. 32. The method of claim 31,
Wherein the first analyte is glucose and the second analyte is hydroxybutyrate. 46. The method of claim 45,
Wherein the dry detection reagent is the dry detection reagent of claim 20, wherein the concentration or presence of one or more analytes is measured electrochemically. 32. The method of claim 31,
Wherein the first analyte and the second analyte are the same, wherein the concentration or presence of one or more analytes is measured electrochemically. 49. The method of claim 47,
Wherein the analyte is glucose, wherein the concentration or presence of one or more analytes is measured electrochemically. 49. The method of claim 48,
Wherein the dry detection reagent is the dry detection reagent of claim 21, wherein the concentration or presence of one or more analytes is determined electrochemically.

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